In this article we will discuss about the process of gastrulation in amphioxus and amphibians.
Gastrulation in Amphioxus:
In Amphioxus, there are differences in the various regions of the egg cytoplasm which permitted Conklin (1932) to trace these regions into the later stages of development and thus to reconstruct a fate map, at least in rough outlines.
At the beginning of cleavage three regions can be distinguished in the Amphioxus egg. Near the vegetal pole a mass of cytoplasm is found which contains the greatest amount of yolk (although yolk in this case is not abundant and the yolk granules are relatively very small). The animal hemisphere of the egg consists of cytoplasm that has less yolk and is consequently more transparent.
On one side of the egg, in a position roughly corresponding to the marginal zone of the amphibian egg, there is a special type of cytoplasm; it does not contain much yolk, but it can be distinguished from the animal cytoplasm by its ability to be deeply stained by basic dyes. The mass of cytoplasm of this kind has a crescentic shape, the attenuated ends of the crescent being drawn out along the equator of the egg about halfway around.
During the period of cleavage the three regions become subdivided into blastomeres without the cytoplasmic substances having been displaced to any great extent. The distinctions which could be traced in the cytoplasm of the egg now become accentuated by further distinctions in the size and shape of the blastomeres.
The vegetal material is now contained in a number of rather large cells taking up the position on and around the vegetal pole of the blastula. The animal hemisphere is made up of cells containing the clear cytoplasm. The cells are columnar and form a very closely packed columnar epithelium.
The cells containing the basophilic cytoplasm are clearly discernible even as to shape. They are the smallest cells in the blastula, even smaller than the animal cells, and they are rather loosely packed, the external surfaces bulging out, as is usually found in the earlier cleavage stages.
The fate of the three regions is the following- The clear cytoplasm that later becomes the animal hemisphere of the blastula develops mainly into skin epidermis. The granular cytoplasm, which takes up the region around the vegetal pole of the blastula, develops into the lining of the alimentary canal. The crescent of basophilic cytoplasm is the material which gives rise to the muscles and the lining of the body cavity and thus represents a mesodermal area.
More recently, the method of local vital staining has been applied to the study of Amphioxus development. It was found that the presumptive mesodermal area is not restricted to one half of the egg only but reaches farther around the equator.
On the opposite side a zone giving rise to the notochord could be detected, and above that a crescentic area which develops into the nervous system. The similarity between this fate map and that of the amphibians is practically complete. There is also a striking similarity to the distribution of different kinds of cytoplasm in the ascidian embryo.
As a result of cleavage in Amphioxus, a ‘blastula is formed which has a large blastocoele and a blastoderm consisting of a single layer of columnar cells. The cells at the vegetal pole are somewhat larger than at the animal pole, and the blastoderm therefore is thicker.
The various cytoplasmic substances present in the egg suffer no appreciable displacement during cleavage, and the fate map presented can equally well be applied to the blastula. The necessity of displacement of the parts so as to put them in the positions where they are situated in the adult animal is amply evident.
Gastrulation is initiated when the blastoderm at the vegetal pole becomes flat and subsequently bends inward, so that the whole embryo, instead of being spherical, becomes converted into a cup-shaped structure with a large cavity in open communication with the exterior on the side that was originally the vegetal pole of the embryo.
The cup has a double wall, an external one and an internal one, the latter lining the newly formed cavity. The external and internal epithelial layers are continuous with each other over the rim of the cup-shaped embryo. In this stage there is still a space between the external and internal walls representing the remnants of what was the blastocoele of the blastula.
The external lining consists of presumptive epidermis and presumptive nervous system. In other words, it consists of parts which have been classified as ectoderm. The internal lining consists mainly of the presumptive gut material, that is, of endoderm.
The presumptive material of the notochord and the-mesodermal crescent at first lie on the rim of the cup but very soon they shift inward so as to occupy a position on the internal wall of the cup. In this way the endoderm, the mesoderm, and the notochord disappear from the surface of the embryo into the interior where they belong. The external surface of the embryo now consists of ectoderm.
The embryo in this stage of development is called a gastrula. The movements of enfolding or inward bending of the endoderm and mesoderm are known as invagination. The cavity arising through the invagination of the endoderm and mesoderm is called the primary gut or archenteron. The opening of the archenteron to the exterior is called the blastopore.
At the same time, the blastopore denotes the pathway by which the endoderm and mesoderm pass into the interior of the embryo. The blastopore, being the opening leading into the primary gut, has been likened to a mouth; its rims, therefore, are usually referred to as the lips of the blastopore. We may distinguish the dorsal lip, the ventral lip, and the lateral lips of the blastopore, respectively.
The blastopore is very broad in the initial stage of gastrulation, but soon the lips of the blastopore begin to contract, so that the opening which leads into the archenteron becomes smaller and is eventually reduced to an insignificant fraction of the original orifice.
This contraction of the lips of the blastopore is connected with the disappearance of the mesodermal crescent material and the presumptive notochord from the rim of the cup-shaped embryo. As more material is shifted into the interior of the gastrula, the remnants of the blastocoele become completely obliterated by the two walls of the embryo coming in contact with each other.
As the presumptive notochord and the mesodermal crescent shift into the interior of the gastrula, they also change their position relative to each other. In the blastula, these two areas lie on opposite sides of the embryo.
Now the lateral horns of the mesodermal crescent converge toward the dorsal side of the embryo and come to lie on both sides of the presumptive notochord. In the next stage that follows the contraction of the rim of the blastopore, the embryo becomes elongated in the anteroposterior direction, all the various presumptive areas participating in this elongation.
The elongation of the notochordal and the mesodermal material brings them into still closer contact with each other, the notochordal material shifting backward, in between the two horns of the mesodermal crescent material.
As a result of these movements, the notochordal material becomes stretched into a longitudinal band of cells lying medially in the dorsal inner wall of the gastrula and flanked on both sides by bands of mesodermal cells similarly stretched in a longitudinal direction. The remainder of the lateral, ventral, and anterior parts of the inner wall of the gastrula consists of endodermal cells.
The external wall of the gastrula similarly takes part in the elongation of the embryo. One of the results of this is that the presumptive material of the nervous system becomes stretched into a longitudinal band of cells lying mediodorsally over the notochordal material but being somewhat broader than the latter.
Gastrulation in Amphibians:
The purpose which gastrulation must achieve in the amphibians is the same as in Amphioxus; the single-layered embryo has to be converted into a stratified structure, with the groups of cells destined to become gut, notochord, muscle, and so on, placed in the interior of the embryo and surrounded by the cellular layer destined to become the outer covering of the body and the nervous system.
However, in the amphibians this cannot be done by the bending inward of the vegetal region of the blastoderm, as in Amphioxus, because the vegetal wall of the blastula is far too thick and overladen with yolk and is therefore not capable of being bent inward as a whole.
The processes leading to the disappearance of the material for the internal organs from the surface are therefore carried out mainly by the more active cells of the marginal zone. A correct understanding of amphibian gastrulation has been achieved by the method of localized vital staining—by applying marks of vital stain on the surface of the embryo and tracing the movements of the stained areas in the course of gastrulation.
The first trace of gastrulation which can be observed in an egg of a newt or a frog is a contraction of the vegetal field. In species in which there is a marked difference in color between a darkly pigmented animal hemisphere and a non-pigmented or lightly pigmented vegetal hemisphere, it can be noted that the light colored area is becoming smaller, and the dark-colored parts are reaching further down below the equator.
The marginal zone, including the gray crescent, which originally is situated around the equator of the embryo, now shifts to a position clearly below the equator. The mass of yolky cells of the vegetal hemisphere starts bulging into the blastocoele; thus, the “floor” of the blastocoele, instead of being concaves, becomes convex. In this way the excess of yolky material due to contraction at the surface is accommodated in the interior of the embryo.
The increase of the surface covered by the darkly pigmented cells is achieved by a stretching and thinning out of the layer of cells constituting originally the animal hemisphere of the blastula. In urodeles the blastula wall of the animal hemisphere tends to become a single layer of columnar cells, but in frogs even after the thinning out it is still a stratified, multilayered epithelium.
Inasmuch as the general shape of the embryo during this initial stage is not changed, and the relative positions of the various parts of the embryo are not altered, this initial phase may be described as the pre-gastrulation movements (of parts of the embryo).
The next step in the process is the appearance of the rudiment of the blastopore, which in Amphioxus is the opening leading into the archenteron. In amphibians the first trace of the blastopore is a concentration of pigment in an area between the marginal zone and the vegetal field, on the future dorsal side of the embryo.
This is due to a further contraction of the surface, brought about by a change of shape of the cells at the border between the vegetal field and the marginal zone. Instead of being cuboid or slightly columnar, the cells of the blastopore rudiment become very elongated, with the main part of the cell body with the nucleus drawing away from the surface, while a harrow elongated distal part of the cell continues to reach the surface of the embryo.
The cells thus become bottle-shaped. The vegetal field, although more lightly colored than the animal hemisphere, is not entirely devoid of pigment, and the granules of pigment are, as in other parts of the embryo, situated mainly in the outermost parts of the bottle-shaped cells. Thus, when the outer parts of the cells become narrow, the pigment granules are concentrated in a smaller area, and this concentration of pigment is noticeable on inspection of the embryo viewed from the outside.
The pigment concentration takes place along the border between the vegetal field and the marginal zone, and is therefore elongated in a transverse direction. It is somewhat diffuse to begin with, but fairly soon, with further concentration, becomes a sharply defined transverse line.
The transformation of the cells in the blastopore region from cuboid or columnar into’ ‘bottle” cells is an expression of a shift of the cell bodies away from the surface, and in the direction of the interior of the embryo. This shift is an active process, an act of movement by the cells concerned.
The attenuated “necks” of the bottle cells, inasmuch as they are attached to each other and jointly constitute the surface of the embryo in the region of the future blastopore, exercise a pull at the surface and eventually cause the surface to cave in.
A shallow pit is thus formed on the surface of the embryo. The pit soon becomes drawn out transversely into a groove, neatly separating the vegetal field from the marginal zone on the dorsal side of the embryo, on the side earlier marked by the gray crescent.
In the next phase of gastrulation, the groove begins to spread transversely, and its lateral ends are prolonged all along the boundary between the marginal zone and the vegetal region until they meet at the opposite, ventral, side of the embryo, thus encircling the vegetal region.
The groove represents the blastopore; the rim of the groove on the upper side (the side nearer to the animal pole) is called the “lip” of the blastopore. The term “lip” is used because the blastopore is supposed to be the “primitive mouth” of the embryo. At the time of its first appearance there is only a “dorsal” lip of the blastopore present, as the blastopore appears on the dorsal side of the embryo.
As the groove spreads out transversely, first the “lateral lips” of the blastopore are formed, and eventually also the “ventral lip”. While the rim of the blastopore is extended to a complete circle, it also shifts over the surface of the vegetal region, moving away from the animal pole and toward the vegetal pole.
By the time the blastopore has the form of a ring, the cells of the vegetal region may still be seen filling the space enclosed by the lips of the blastopore. These cells are then called the yolk plug. The rim of the blastopore, however, continues to contract and at last covers the yolk plug altogether.
In this way the material of the vegetal region disappears eventually into the interior of the embryo. When the blastopore contracts to such a degree that the yolk plug disappears from view, the blastopore is said to be “closed.” This term is not quite exact, however, since a narrow canal leading into the interior still persists.
If the blastoderm above the rim of the blastopore is marked with spots of vital stain, it can easily be seen that the stained areas stretch toward the lips of the blastopore, approach its rim, roll over the edge, and disappear inside. Once inside, the stained material does not come to rest but continues its movements in the interior of the embryo, but this time it moves away from the blastopore in the opposite direction from that which it followed while it was still on the surface of the embryo.
Also, the movement of the superficial material can be seen to be most rapid and extensive on the dorsal meridian of the embryo. More material is invaginated over the dorsal lip of the blastopore than over the lateral lips and the least over the ventral lip of the blastopore.
This accounts for the varying breadth of the marginal zone around the circumference of the egg. In fact, the upper limit of the marginal zone is none other than the limit to which the blastoderm is invaginated during the process of gastrulation. The vegetal region is similarly that part of the blastoderm which is enclosed by the rim of the blastopore.
The animal region is the part of the blastoderm that does not pass into the interior by way of the blastopore. The extent of the invagination can be clearly seen if a series of vital stain spots is made along the mid-dorsal meridian of the embryo.
The movement of the cellular material of the embryo in the blastopore area toward the interior also can be observed if the embryo is dissected to reveal the blastocoele cavity. It may be noticed then that on the dorsal side of the embryo the floor of the blastocoele becomes raised, and a broad tongue-like mass of cells is seen rising against the inner surface of the blastoderm.
In the early stages of its formation this mass of cells is very loose and does not surround a cavity. Somewhat later, however, the pit or groove at the surface becomes deeper and is extended in the direction of the mass of cells moving upward into the blastocoele cavity.
On the dorsal side of the embryo, where the groove first appeared and where the streaming of cells into the interior of the embryo is the most active, a cavity is soon formed leading from the groove on the surface into the interior of the embryo. This cavity is lined on all sides by the invaginated cells and represents the archenteron. The archenteron is at first a narrow slitlike cavity.
Then at about the time when the blastopore, as seen from the outside, becomes ring-shaped and surrounds a yolk plug, it is seen that as more material becomes invaginated the archenteron expands at its anterior end and in so doing encroaches on the blastocoele. The latter is eventually obliterated. The lining of this expanded cavity becomes quite thin.
The part of the archenteron adjoining the exterior, and surrounding the yolk plug, remains slit-like right to the end. While the rim of the blastopore moves over the surface of the vegetal region, the vegetal region is drawn into the interior and at the same time is caused to rotate, so that after the end of gastrulation it comes to lie in the ventral part of the archenteron, its originally exterior surface facing its cavity. The opposite, dorsal, wall of the archenteron consists of cells of the marginal zone which have rolled into the interior over the dorsal lip of the blastopore.
The material of the animal region, including the presumptive epidermis and the presumptive nervous system, greatly increases its surface during gastrulation, and at the end of gastrulation it covers the whole embryo after the mesoderm and the endoderm have disappeared inside.
The expansion of the ectoderm is an active process, and the increase of surface area proceeds at the expense of a thinning out of the epithelial layer. The presumptive epidermis expands in all directions, but in the case of the presumptive nervous system the expansion is mainly in the longitudinal direction, i.e., toward the blastopore.
In the transverse direction, on the other hand, the presumptive nervous system area contracts, and the material of the lateral horns of the crescentic area is drawn in toward the dorsal side of the embryo. As a result, the whole nervous system area changes its shape and becomes oval, elongated in an anteroposterior direction. The spreading of the ectoderm to cover other parts of the embryo is a case of so-called epiboly.
The movement of parts of the marginal zone into the interior of the embryo is very different from the invagination as seen in Amphioxus. Only during the formation of the original pit or groove is there a true bending inward of a portion of the blastoderm.
Once the depression has been formed, the further movement inward can be better described as a rolling of superficial cells over the rim of the blastopore into the interior, while new portions of the blastoderm approach the rim in their stead.
This movement has also been described as “involution”. The notochord rolls over the dorsal lip of the blastopore into the interior of the embryo and becomes stretched along the dorsal side of the archenteron, forming the mid-dorsal strip of the archenteron roof.
As it does so, the presumptive notochord undergoes a very considerable elongation in the longitudinal direction and a corresponding contraction in the transverse direction. The notochordal material becomes concentrated on the dorsal side of the embryo, as is the presumptive neural system, but to a much greater degree.
The Prechordal Plate:
The prechordal plate, which in the blastula lies just below the presumptive notochord, is the first mesoderm to invaginate and does so by rolling over the dorsal lip of the blastopore and becoming a part of the archenteron roof in front of the anterior end of the notochordal material.
Of all parts of the blastula, the mesodermal area undergoes the most complicated movements. Most of the mesoderm invaginates into the interior by rolling over the lateral and ventral lips of the blastopore. Once inside the embryo, the mesoderm moves in an anterior direction as a sheet of cells, penetrating between the ectoderm on the outside and the endoderm on the inside.
The mesoderm in Urodela detaches itself from the endoderm and moves forward between the ectoderm and the endoderm, having a free edge anteriorly but preserving an un-interrupted connection with the notochordal material on the dorsal side of the embryo.
The notochordal and the mesodermal materials in this stage are in the form of one continuous epithelial sheet, the chordomesodermal mantle. In the Anura, the mesoderm does not split off from the adjoining endoderm until gastrulation is nearly finished. The result is, however, the same – The formation of the chordomesodermal mantle lying between the ectoderm and the endoderm.
As the mesoderm moves from the posterior end of the embryo (represented by the blastopore) toward the anterior end, there remains at the anterior end a region which the mesodermal mantle has not yet reached. This region, which is free of mesoderm, diminishes as gastrulation proceeds but does not disappear completely. It is in this mesoderm-free region at the anterior end of the embryo that the mouth is later formed.
The concentration toward the dorsal side of the embryo, noted in respect to the notochordal and nervous system material, is also very distinct in the case of the mesodermal mantle. The movement of the mesoderm inside the embryo does not follow the same path as on the surface. If the trajectories of all parts of the presumptive mesoderm are traced, they are seen to converge toward the dorsal side where they represent the movement of the mesoderm after its invagination.
As a result, the mesodermal material becomes concentrated toward the dorsal side. The mesodermal layer is thickest in the roof of the archenteron, where the mesoderm adjoins the notochord; it is thinned out in the lateral part and still more so in the ventral part of the embryo. The mesoderm continues to invaginate by rolling over the rim of the blastopore, even after the endoderm has come to lie inside and the yolk plug has disappeared from the surface.
The invagination of the mesoderm may therefore be considered as retarded when compared with the invagination of the endoderm. The degree of retardation varies in different animals. It is the least in frogs, greater in the urodeles, and greatest in the lamprey whose gastrulation is, otherwise, similar to that of the amphibians. As a result of the retardation, the presumptive mesoderm of the tail region and sometimes also the presumptive mesoderm of the posterior trunk region may still be on the surface of the embryo when the blastopore is “closed.”
The presumptive endoderm is found, in the blastula, partly in the marginal zone and partly in the vegetal region. The two parts of the presumptive endoderm invaginate in different manners; the part lying in the marginal zone is mainly absorbed into the original pit-like invagination of the blastopore, and the vegetal region disappears from the surface when it becomes covered up by the contracting rim of the blastopore.
The blastopore first appears as a pit in the endodermal area, between the marginal zone endoderm and the vegetal endoderm. The endodermal cells lying at the bottom of the pit are later found in the duodenal region of the embryo. In the early gastrula the presumptive endoderm of the pharyngeal and oral region lies on the anterior slope of the pit and is therefore invaginated as part of the dorsal lip of the blastopore.
This material later forms the most anterior part of the advancing archenteron. In the later stages of gastrulation, the oral and pharyngeal endoderm expands so as to form the spacious foregut, whose lateral, ventral, and anterior walls then consist of a rather thin layer of endoderm. Only part of the dorsal wall of the foregut is taken up by the prechordal plate and the anterior tip of the notochord.
The endoderm of the vegetal region passes into the interior of the embryo more or less passively and there comes to lie in the floor of the archenteron. It is not pushed forward as far as the marginal zone endoderm, but remains confined to the middle and posterior parts.
The layer of endoderm in the floor of the archenteron is very thick, and the cavity of the archenteron is therefore reduced posteriorly to a rather narrow canal underneath the chordomesodermal mantle. This narrow part of the archenteron later becomes the mid-gut of the embryo. The lateral endodermal walls of the mid-gut are much thinner, and dorsally they end at a free edge after the mesoderm has been split off from the endoderm.
Gastrulation in the Anura:
The description of amphibian gastrulation applies, strictly speaking, to the urodeles—newts and salamanders. Quite considerable deviations from the pattern of urodele gastrulation are found to occur in frogs and toads. Some of these deviations may be connected with a relatively greater amount of yolk in frog eggs, even if the actual size of the egg is smaller.
The fate map for the frog embryo at the stage just before gastrulation, drawn up on the basis of vital staining experiments, shows the marginal zone (the part that becomes invaginated into the interior), as well as the area destined to form the nervous system, as being narrower, so that the neural system area does not reach to the animal pole, as it does in urodele embryos.
In frogs the mesoderm remains in close contact with the endoderm during invagination, and the two separate from one another at a later stage.
The greatest deviation from what was revealed by the study of development in newts has been found in the clawed frog, Xenopus—a species much used for experiments in recent years because of the ease with which this animal can be kept in captivity and the possibility of obtaining eggs at all times by hormonal injections.
It has been found that in Xenopus in the blastula stage the whole of the presumptive chordomesoderm is located in the “inner marginal zone.” The cells forming the superficial layer of the multilayered blastoderm give rise either to the skin epithelium and nervous system, or to the lining of the endodermal gut.
When the surface layer of the marginal zone is drawn into the interior over the dorsal lip of the blastopore, it forms the endodermal lining of the roof of the gut, so that the cavity of the gut, from its earliest formation, is lined by the endoderm only.
The cells of the presumptive notochord, which in the blastula lie in the inner marginal zone, accompany the endodermal cells in their forward thrust (away from the rim of the blastopore and toward the animal pole) but at no stage form part of the dorsal lining of the archenteron, and also at no stage are they on the exterior surface of the embryo.
Consequently there need be no secondary fusion of edges of the gut endoderm to enclose the gut cavity, as described for Amphioxus and for urodele amphibians. The mesoderm likewise advances from the blastopore rim together with the endoderm, at all stages, underneath the surface layer of the embryo, and not encroaching on the cavity of the gut.
Even in urodeles part of the mesoderm (presumptive heart, blood islands, and also parts of the somite’s) are produced from cells of the inner marginal zone; that is, from cells never exposed to the surface of the embryo. Whatever the position of the presumptive notochord and mesoderm in the blastula, and their mode of attaining their final position in the embryo, as result of gastrulation they form a continuous layer between the ectoderm and endoderm – the chordomesodermal mantle. The process of gastrulation in other frogs and toads appears to be intermediate between that found in urodeles and that of Xenopus.