Book - The Frog Its Reproduction and Development 7
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Rugh R. Book - The Frog Its Reproduction and Development. (1951) The Blakiston Company.
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Chapter 7 - Gastrulation
Gastrulation is that dynamic process in early development which invariably results in the transformation of a single-layered blastula stage to a didermic or 2-layered embryo. It involves, but is independent of, mitosis. The process varies considerably among the Vertebrates, but to a lesser extent when the process is compared in closely related species. The two layers to be distinguished are the ectoderm and the endoderm. In many forms (e.g., the frog) the third germ layer, or mesoderm, is formed almost simultaneously with the endoderm.
Significance of the Germ Layers
The distinction of germ layers, such as the ectoderm, mesoderm, and endoderm, is purely a matter of human convenience and is of no real concern to the embryo. The mere fact that the endoderm and the mesoderm are both derived originally from the ectoblast (outermost layer of the blastula) suggests their fundamental similarity. By means of experimental procedures it is possible to demonstrate that these presumptive germ layers are interchangeable and that regions ordinarily destined to become, for example, brain (ectoderm), may be transplanted to another region of the blastula where they will become muscle (mesoderm) or possibly thyroid (endoderm). The germ layer distinctions are based upon their position in the developing organism, and their fate. The ultimate tissues of the adult are often classified, again as a matter of human convenience, on the basis of groups which are derived from one or another of these three primary germ layers. The germ layers, which are first distinguishable with gastrulation, therefore are definable by their fate in embryonic development. However, this fate is an arbitrary distinction since the fate of any group of cells can be altered by transplantation. The importance, then, of this distinction is one of position, and the ectoderm is always the outermost layer of cells, the endoderm the innermost, and the mesoderm the intermediate layer of cells present when gastrulation has been completed.
In recapitulation, a histologist could define the ectoderm as that group of embryonic cells which, under normal conditions of development, give rise to the epidermis and the epidermal structures, to the entire nervous system and sense organs, to the stomodeum, and to the proctodeum. The endoderm would be defined as that group of embryonic cells which normally give rise to the lining epithelium of the entire alimentary tract and all of its outgrowths, such as the thyroid, lungs, liver, pancreas, etc. The mesoderm would be defined as that group of cells which normally give rise to the skeleton and connective tissues, muscles, blood and vascular systems, to the coelomic epithelium and its derivatives, and to most of the urogenital system. The notochord in the frog is derived simultaneously with the mesoderm, and probably from the dorsal lip epiblast.
It is important, therefore, for the student to avoid ascribing any peculiar powers to the germ layers as such. They have their particular fate in development not by virtue of any peculiarly endowed power but rather by their position in the developing embryo as a whole.
Origin of Fate Maps
Gastrulation is a critical stage in the development of any embryo. This is due in part to the fact that the positional relationship of the various cells of the late blastula and early gastrula begins to take on special significance. This has been demonstrated by many experimental embryologists, among whom are His, Born, BUtschli, Rhumbler, Spek, Vogt, Dalcq, Pasteels, Vintemberger, Holtfreter, Nicholas, and Schechtman. They have used various experimental devices, such as injury or excision of cell areas, or staining local areas with vital dyes and following their subsequent movement. By such methods the socalled "fate maps" of various blastulae have been worked out.
A fate map is simply a topographical surface mapping of the blastula with respect to the ultimate fate of the various areas. When one traces a cell group on the surface of a blastula which has been vitally stained with Nile blue sulfate, for instance, and finds that these cells move from the marginal zone (between the animal and the vegetal hemispheres) over the dorsal lip of the early blastopore and into the embryo where they become pharyngeal endoderm, he is then able to label the fate of that area on other blastulae. The fate maps of various amphibia are basically alike, but they are not sufficiently alike in detail to permit plotting a universal amphibian fate map. The frog's egg is very dark and it is difficult to apply vital dyes so that they will be visible on the frog blastula. However, the fate maps of closely related species have been worked out and, combined with circumstantial experimental evidence on the frog's egg, we are able to supply what is believed to be a reasonably accurate fate map of the frog blastula.
Under conditions of normal development, the ectoderm of the frog is derived in part from the cells of the animal hemisphere and in part from the intermediate or equatorial plate cells. These are the regions on the fate map designated as presumptive ectoderm. The endoderm comes partly from the intermediate zone but largely from the vegetal hemisphere area (i.e., the presumptive endoderm on the fate map). The mesoderm and the notochord have a dual origin, arising between the ectoderm and the endoderm, largely from the region known as the lips of the blastopore. The student is advised to study carefully the accompanying fate maps.
Gastrulation as a Critical Stage in Development
One of the reasons for the fact that gastrulation is such a critical stage in embryonic development is that not only do the cells divide but also various cell areas take on special significance which they did not previously have. This special significance is shown by the mere fact that fate maps have been derived. There is a new independence, as well as interdependence, of various areas of the embryo. This change is designated as differentiation. Instead of a group of somewhat similar cells, arranged more or less in a sphere, each cell having essentially the same potentialities as any other cell, there is now a mosaic of cell groups of integrated differences. The differences in the various areas may not be apparent physically, but they can be demonstrated functionally and constitute the process of differentiation. This is probably the most critical change that is brought about by gastrulation.
Another reason for the critical nature of gastrulation lies in the mechanics of the process itself. There is a localized interruption in the , continuity of the epibolic movement of the surface coat toward the vegetal hemisphere. This is only an apparent interruption, since it involves an inturning of cells at a very specific region of the marginal zone, or the most ventral limit of the pigmented animal hemisphere cells. This specific region is the ventral limit of the original gray crescent, destined (at the time of fertilization) to become the region of formation of the dorsal lip of the blastopore, with all of its implications.
Cells which lie on the lateral surface of the late blastula begin to roll inwardly, first only a few cells and then, by a sort of contagion, the contiguous cells of the more lateral marginal zone. It is this infolding process which marks the actual, observable process of gastrulation. It must be emphasized that these observable changes are probably long anticipated, as will be suggested by the detailed description below. If there is interference with this inturning movement of cells, any subsequent development is apt to be abnormal or incomplete if, in fact, it occurs at all. Embryos at the time of gastrulation are indeed hypersensitive to physical changes in the environment, and to genetic incompatibilities within the chromosomes of the involuting cells. Embryos which survive this process may reasonably be expected to achieve the next major step, namely neurulation.
There is no clear-cut demarcation between the blastula and the gastrula stages, unless one accepts the initial involution of the dorsal lip cells. Some investigators have pointed out that there is a disproportionate ratio of the yolk and cytoplasm to the nucleus of the fertilized egg, as compared with the ratio in the somatic cell. This suggests to them that cleavage results in a progressive approach to the somatic ratio of nuclear volume to cytoplasmic volume. When the ratio is reached whereby the nucleus can properly control its cytoplasmic sphere of influence, then the latent influence can begin to exert itself and the process of differentiation begins. Such a situation occurs at the beginning of the gastrula stage. Cleavage continues, of course, but it has been proved beyond a doubt that cell areas are no longer of equivalent potency with regard to ultimate development. It has been suggested, therefore, that the blastula stage is monodermic (one principal outer layer of cells, generally arranged somewhat spherically) and contains a blastocoel. The end of the blastula stage is reached when the nuclear to cytoplasmic volume relationship of the constituent cells becomes most efficient, differentiation begins, and we can observe the movements and surface changes attendant upon gastrulation.
Until recently the yolk hemisphere of the early cleavage stages of the amphibian embryo was considered to be relatively inert. It was even considered a deterrent to morphogenetic movements of gastrulation. The vegetal hemisphere becomes cellular, but more slowly than does the animal hemisphere. The cells are larger and always contain abundant yolk. Nicholas (1945) wrote: "The concept of the inertness of the yolk mass probably inhibited our realization of its possible import." This attitude is now subject to change.
By applying vital dyes to the yolk hemisphere of the early and late blastula stages, Nicholas has found positive activity on the part of these yolk-laden vegetal pole cells, suggesting that they are concerned with the formation of the blastocoel, with the process of ingression, and also with the changes in water balance and later rotation. It now seems that the yolk assumes a dynamic rather than a passive role both in the process of blastulation and in anticipating the changes prerequisite to gastrulation.
In an exhaustive series of studies, Holtfreter and Schechtman have attempted independently to analyze the morphogenetic movements both before and during the gastrulation process in the amphibia. It is very probable that the formative influences so evident at the time of gastrulation are present long before the initial involution of cells to form the dorsal lip of the blastopore. The following description is based largely on the studies of these investigators and is supported by direct observation, available to anyone.
There is a protein-like surface coating or film on amphibian eggs, present from the beginning. It has plastic elasticity, is not sticky on its outer surface, but is an integral syncytial part of the living egg. This coat persists and its strength increases during development. It is involved in matters of cellular elasticity, cell aggregation, cell polarity, cell permeability, resistance to external media, osmotic regulation, and tissue affinity — all physical phenomena which are important in gastrulation movements.
This surface coating is divided only superficially by the segmenting blastomeres of the early embryo and yet (Holtfreter): "the syncytial coat thus represents at least one, and perhaps the most significant one.
of the effective forces that make for a morphogenetic integration of the dynamic functions of the single cells into a cooperative unity." Embryologists have long been in search of a controlling supercellular physical force which might explain at least the initiation of the movements leading to gastrulation. This surface coat of Holtfreter may well be that controlling and integrating force. He suggests further that probably all of the non-adhesive epithelial linings of the larva can be traced back to this original surface coat.
The blastula stage is held to a spherical aggregate by the presence of this surface coating. However, since the coating is not itself divided with each cleavage, but simply folds into the furrows between blastomeres, the inner boundaries of the cells do not have the same dominating force that is present on the outer surface. Such cells are found to be interconnected by slender and temporary processes. The formation of cells, once thought to be the cause of gastrular movements, is now thought to be merely a convenience for the execution of such movements.
Epiboly in the frog is the concern of this surface coat rather than of individual cells. It is due to the expansive nature of the surface coating while it is in contact with a suitable substratum. The potential ectoderm (animal hemisphere cells) and the potential endoderm (vegetal hemisphere cells) are, in fact, competitors in the tendency to cover the surface area. The driving force is this surface coat, and for some reason the potential of the animal hemisphere portion of it is greater than the potential of the surface coat of the vegetal hemisphere.
Definition of the Major Processes of Gastrulation
Gastrulation is now recognized as an extremely complicated but highly integrated and dynamic change in the embryo, brought about by a combination of physical and chemical forces arising intrinsically but subject to extrinsic factors. We do not yet fully understand the process in any animal, particularly because of the elusive nature of the forces involved. Gastrulation is concerned with cell movements, changes in physical tension, and in the metabolism of carbohydrates, proteins, and possibly even the lipids.
Before attempting to describe the process as it occurs in the frog, it would be well for us to have a clear-cut understanding of the meaning of the various terms often encountered in such a description. The student is advised to consult frequently the Glossary at the end of this text, not only while reading this section but also throughout the discussion of embryology as a scientific subject.
Invagination — As used by Vogt, this term means insinking (Einstulping in German) of the egg surface followed by the forward migration (Vordringen) which involves the displacement of inner materials. Schechtman uses the term to mean an inward movement, without any reference to whether there is pulling, pushing, or autonomous movement. There is probably some invagination in gastrulation of the frog's egg.
Involution — As used by Vogt, this term means a turning inward, a rotation of material upon itself so that the movement is directed toward the interior of the egg. Involution does occur in the frog's egg gastrulation.
Delamination — This is a splitting-oflf process whereby the outer layer of ectoderm gives rise to an inner sheet of cells known as the endoderm. It does not occur in the frog's egg gastrulation.
Epiboly — This is a progressive extension of the cortical layer of the animal hemisphere toward the vegetal hemisphere, or a sort of expansion from animal toward the vegetal hemisphere. In some eggs (e.g., the mollusk Crepidula) it actually involves an overgrowth by smaller cells over the larger vegetal hemisphere cells. The extension type of epiboly does occur in the frog's egg gastrulation.
Extension — This term is used by Schechtman to describe in amphibia the self-stretching process which seems intrinsic within certain cell areas, particularly in the dorsal region of the marginal zone. It is
Constriction — This term is used to describe the convergence of cell areas resulting in the gradual closure of the blastopore. It is due to a narrowing of the marginal zone and a pull, or tension, of the dorsal lip. This occurs in the frog's egg gastrulation.
Dorsal convergence — This is the dorsal Raffung of Vogt, used to describe the movement of the marginal zone cell areas toward the dorsal mid-line during involution and invagination.
With this general introduction to a very fascinating but, as yet, little understood phase of embryological development, we shall retrace the steps to the late blastula stage and endeavor to anticipate the process of gastrulation in the frog.
To understand the processes to be described as gastrulation, let us list in succession the changes that may be observed in the frog embryo.
- Thinning of the gray crescent side of the blastula wall. There appears to be an actual migration of cells from the deeper layers of the blastula away from the original site of the gray crescent. This is the region where involution will first take place, the region which will come to be known as the dorsal lip of the blastopore. The blastula wall which is equatorially opposite to the gray crescent side may, in consequence, become several cells thicker.
- Continued epiboly or extension of the marginal cell zone toward the vegetal hemisphere , so that the diameter of the marginal zone becomes progressively smaller as it passes below the equator. The marginal zone attains increasing rigidity so that it exerts an inward pressure on the yolk cells which are being encircled, causing them to be arched upward toward the blastocoel. One might draw the simile of an overlapping rubber membrane being drawn down over a mass of slightly less resistant material, toward the center of which there is a cavity. The yolk endoderm cells toward the gray crescent side become separated from the epiblast and are forced upward, displacing the blastocoel and reducing its size. This process is known as pseudoinvagination because there is a degree of "pushing in" of the vegetal hemisphere cells. It is not true invagination, however, as one understands the process in such a form as the starfish embryo. The slit-like space between the yolk endoderm and the epiblast, continuous with the blastocoel, is sometimes referred to as the gastrular slit.
- The initial involution or inturning of a jew cells at the lower margin of the original gray crescent, followed by the lateral extension of this involution along the epibolic marginal zone. This region of initial involution becomes the dorsal lip of the blastopore. The marginal zone cells become separated from the more ventral and lighter colored yolk cells. The first cells to involute do not lose continuity with their neighbors, but carry with them the adjacent, contiguous lateral cells of the marginal zone. The infolding or inturning process therefore begins at one point but is continued around the marginal zone.
- Further epiboly of the entire marginal zone toward the vegetal hemisphere, apparently interrupted only at the levels of involution. The inturning cells are rolling under themselves like an oncoming wave. The margin of the wave continues, through epiboly, to move toward the vegetal hemisphere but at a rate which seems slower than that of the marginal zone which has not yet involuted (i.e., opposite the dorsal lip cells). In other words, cells are moving inwardly over the dorsal lip of the blastopore, but the margin of the lip itself is moving vegetally as a result of epibolic extension. Some epibolic movement is absorbed in involution.
- The piling up or confluence of animal hemisphere cells, many of which are destined to move inside over the blastoporal lips.
- The continued lateral extension of the involuting marginal zone cells so that there is eventually a circumferential meeting of the blastoporal lips. These comprise the dorsal, lateral, and ventral lips of the blastopore, not clearly demarked. The vegetal hemisphere cells, which are now exposed within a ring of involuted marginal zone (lip) cells, are collectively known as the "yolk plug." The term yolk plug is used because the cells of the pig merited marginal zone (blastoporal lips) are in sharp contrast with the surrounded vegetal pole yolk-laden cells.
- The future epiholy of the marginal zone, accompanying invohition at all points, resulting in a continued reduction in the size of the circumblastoporal lips and of the exposed yolk plug. The margins of the yolk plug are at first round, then vertically oval, and finally the lateral lips of the blastopore approach each other to form a vertical slit as the yolk plug is closed over and disappears within.
- The origin of the internal or second layer of cells, arising from the involuted dorsal lip cells and collectively known as the endoderm. This sheet of cells fans out within the embryo to give rise very soon to a new cavity, the gastrocoel or archenteron. Since these inturning cells give rise largely to the roof and lateral walls of the archenteron, those parts of the cavity will be lined with somewhat pigmented cells from the original epiblast. There is a gradation or lessening of this pigment in the archenteric roof cells as one progresses anteriorly in the gastrula.
- Simultaneously with the origin of the inner layer of endoderm, some cells are proliferated o§ into the gastrular slit, between the roof of the archenteron and the dorsal epiblast, which cells will become the notochord. Before differentiation these cells are called chorda-mesoderm. Since the lips of the blastopore are circular, this proliferated mass of cells also becomes circular. The more lateral and also the ventral proliferations become mesoderm. There arises, therefore, the dorsal notochord and the lateral and ventral mesoderm as a circle of tissue within the fold of the blastoporal lips, occupying the space between the epiblast and either the inner endoderm or yolk endoderm.
By this time the student must have realized that gastrulation is a highly integrated mosaic of motions which are purposeful in that they achieve the state of the completed gastrula. Spemann aptly wrote:
- However harmonious the process of motion may be by which the material
for the chief organs arrives at the final place, and however accurately the single movements may fit together . . . they are nevertheless no longer causally connected, at least from the beginning of gastrulation onward. Rather, each part has already previously had impressed upon it in some way or other, direction and limitation of movement. The movements are regulated, not in a coarse mechanical manner, through pressure and pull of simple parts — but they are ordered according to a definite plan. After an exact patterned arrangement, they take their course according to independent formative tendencies which originate in the parts themselves. Thus we find in the gastrula stage a mosaic, a pattern of parts with definite formative tendencies from which the formative tendencies of the whole must necessarily follow.
Gastrulation, according to Spemann, is an intricately arranged mosaic of parts possessing autonomous movement potentials which act in the right way at the right time.
|Blastula to gastrula stages in the frog — Shown by stereograms. (Modified and redrawn from Huettner.)|
The forces which bring about the initial involution or inturning of cells at a specific point of the marginal zone to form the dorsal lip of the blastopore have not yet been identified. The nearest approach to a valid explanation is that of Holtfreter. He says:
- The alkalinity of the blastocoel can be assumed to be strong enough to
establish, in cooperation with the suspended protein particles, an efficient gradient of surface tension between the internal and external medium, as envisaged by Rhumbler. Those cells which are in interfacial contact with both media would be primarily affected by it. They will tend to move in the direction of the surface tension lowering alkaline medium, and to reduce their contact area with the external medium having a higher surface tension. Being, however, attached to the peripheral coat they can only stretch along the gradient, assuming a shape which can be expected to correspond to those claviform cells which we find in all cases where invagination takes place. The peipendicular stretching will have to continue as long as the gradient persists and until the cells have attained a position where their potential energy is lowered to a least possible value. This movement is, however, conditioned by the cellular plasticity which is restricted. Thanks to the tensile strength of the cell wall, the attenuated neck portion is subjected to an increasing mechanical stress. The surface yields and is pulled inside in the form of the archenteron.
Schechtman (1942) calls involution an "insinking" and then explains what follows, after having demonstrated that various areas of the pre-gastrula have various types of autonomous movement. He says:
- Gastrulation begins with autonomous movements; the in-sinking of the presumptive pharyngeal endoderm, the stretching of the marginal zone toward the blastoporal groove, the forward migration of the internally situated marginal material along the underside of the animal hemisphere. As the stretching presumptive chordal region comes to the edge of the blastoporal lip, it is progressively carried under by the invagination and involution of the adjacent portions of the marginal zone. This insures that the presumptive chorda is in a position to exert a double effect by means of its extension, for it will not only pull the lateral marginal zones dorsaiward, but will also carry them forward in a dorsal position. Meanwhile the blastoporal lips are constricted, since there is progressive withdrawal of marginal zone material by the process of dorsaiward convergence somewhat as the mouth of a purse is constricted when the purse string is pulled. The tendency of constriction is augmented further by the forward migration of the internal portions of the marginal zone, for this movement also tends to withdraw material from the region of the blastoporal lips.
In summary, Schechtman believes:
- The presumptive chorda is invaginated by the inwardly directed tension or pull exerted by the invagination and involution of the lateral marginal zone, with which it is continuous.
- The lateral marginal zones are then pulled dorsaiward and inward in the dorsal position by the autonomous stretching and simultaneous narrowing of the presumptive chorda.
- The constriction of the blastoporal lips over the yolk mass is affected by the progressive withdrawal of marginal zone material by dorsaiward convergence.
Internally the involuted cells extend anteriorly, away from the point of involution or the dorsal lip of the blastopore. The inturned endoderm surrounds the new cavity, which is at first no more than a slit. The cavity (archenteron) rapidly expands in all directions so that the blastocoel, originally in a dorsal position, becomes progressively displaced anteriorly or away from the side of the blastopore formation. The blastocoel is later displaced antero-ventrally by this expanding archenteron. It also becomes reduced in size until finally it is found only as the slit between the endoderm and the yolk, referred to as the gastrular slit. Sometimes there is a remnant of the blastocoel, separated from the archenteron by a single layer of cells called the completion bridge. This bridge is of no consequence, and frequently ruptures to merge the contents of the blastocoel and the gastrocoel.
These movements of involuting cells and expanding endoderm result in an enlarged archenteron (gastrocoel), entirely lined with endoderm. As stated, the roof and lateral wall cells are more pigmented than the yolk endoderm floor cells of the archenteron. The opening beneath the dorsal lip of the blastopore, and into the archenteron, is called the blastopore. This is an incorrect term, however, for the pore opens into the gastrocoel and not into the blastocoel. The blastopore is occluded by the yolk endoderm cells but later opens into the perivitelline space by the withdrawal of the yolk plug. It will close ultimately with the development of tail mesoderm.
The reduction of the blastocoel and the enlargement of the archenteron together alter the gravitational axis of the entire embryonic mass, shifting it about 90" away from the position of the blastopore. The yolk plug, instead of remaining in a ventral position, comes to lie about the level of the original equator. This all occurs while the embryo lies free within its fertilization membrane. Further, the presence of the archenteric roof and the chorda-mesoderm cells above it cause the overlying ectoderm to thicken and the whole embryo becomes elongated in an antero-posterior direction, more or less horizontal to the revised center of gravity. The yolk plug now marks the posterior region of the future embryo.
It must be emphasized that, during this shift in the gravitational axis, there has not been a comparable shifting of the internal relationships of the embryo. The blastopore and all related parts of the embryo have been rotated dorsally, within the enveloping fertilization membrane. The axis of the gastrula is simply altered by the development of this new cavity, the archenteron, with its consequent obliteration of the blastocoel.
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Rugh R. Book - The Frog Its Reproduction and Development. (1951) The Blakiston Company.
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