Cleavage: Definition and Patterns | Fertilization | Embryology

In this article we will discuss about:- 1. Definition of Cleavage 2. Chemical Changes during Cleavage 3. Patterns 4. Nuclei of Cleavage Cells 5. Morula and Blastula.

Definition of Cleavage:

One of the peculiarities of sexual reproduction in animals is that the complex multicellular body of the offspring originates from a single cell—the fertilized egg. It is necessary, therefore, that the single cell be transformed into a multicellular body.

This transformation takes place at the very beginning of development and is attained by means of a number of cell divisions following in rapid succession. This series of cell divisions is known as the process of cleavage.

Cleavage can be characterized as that period of development in which:

1. The unicellular fertilized egg is transformed by consecutive mitotic divisions into a multicellular complex.

2. No growth occurs.

3. The general shape of the embryo does not change, except for the formation of a cavity in the interior—the blastocoele.

4. Apart from transformation of cytoplasmic substances into nuclear substance, qualitative changes in the chemical composition of the embryo in cleavage are limited.

5. The constituent parts of the cytoplasm of the egg are not displaced to any great extent and remain on the whole in the same positions as in the egg at the beginning of cleavage.

6. The ratio of nucleus to cytoplasm, very low at the beginning of cleavage, is, at the end, brought to the level found in ordinary somatic cells.

Chemical Changes during Cleavage:

Although there is no growth during the period of cleavage, chemical transforma­tions do occur, and at least some are markedly intensified, as compared with the conditions in the unfertilized egg.

The most obvious change observed during cleavage is a steady increase of nuclear material at the expense of cytoplasm. The number of nuclei is of course doubled with every new division of the blastomeres, and this doubling is accompanied by an increase of nuclear substance, which involves an increase of deoxyribonucleic acid—the amount per nucleus of the latter remaining constant.

The increase in the chromosomal deoxyribonucleic acid, at least during the earlier phases of development, must be at the expense of materials contained in the egg. There are several possible sources of such materials. First, the nucleic acids present in the cytoplasm of the eggs should be mentioned.

In sea urchin eggs there is a large amount of ribonucleic acid in the egg cytoplasm, and this gradually disappears later in develop­ment. When sea urchin embryos are supplied with radioactively tagged uridine, some of it is later incorporated into the DNA.

Such incorporation is made possible by the presence in developing sea urchin eggs of an enzyme, ribonucleotide reductase, which converts ribonucleotides into deoxyribonucleotides. DNA may, however, be synthesized in the cleaving egg directly from low molecular weight precursors. This has been proved by supplying such precursors, labeled with radioactive atoms, to cleaving eggs of sea urchins and amphibians.

When cleaving sea urchin eggs were kept in seawater contain­ing 14C-labeled glycine (which may be used for the synthesis of purine groups in the nucleic acid molecule), it was found that the radioactive carbon atoms were incorpo­rated in large amounts into the deoxyribonucleic acid, bypassing the cytoplasmic ribonucleic acid. Also, when 14C-labeled glycine was injected into fertilized frog eggs, some of it was incorporated into deoxyribonucleic acid.

The second important aspect of metabolism during cleavage is the synthesis of ribonucleic acids, which is believed to be very limited, although not absent altogether. In frogs, ribosomal RNA apparently is not produced at all until after completion of cleavage.

As the nucleolus is the site of synthesis of rRNA, this organelle is completely lacking in these animals during cleavage. It reappears in the nuclei at the onset of gastrulation simultaneously with the resumption of ribosomal RNA synthesis.

In the sea urchin there is very little ribosomal RNA produced during cleavage, but in both the amphibians and sea urchins synthesis increases drastically at the onset of gastrulation. Messenger RNA and transfer RNA, on the other hand, are synthesized during cleavage, or at least in the later stages of cleavage.

Synthesis of RNA, however, does not seem to be necessary for cleavage, since eggs which are treated with actinomycin D and in which presumably DNA dependent RNA synthesis is suppressed continue cleaving normally. It is concluded, therefore, that any messenger RNA produced during cleavage remains inactive or “masked,” similar to the messenger RNA in unfertilized eggs.

Fertilization in sea urchins leads to a spectacular increase in protein synthesis, and this is continued throughout the period of cleavage. In other animals, such as amphibians, protein synthesis does not markedly change after fertilization; a certain amount of protein synthesis, however, takes place throughout the period of cleavage. The amount of active cytoplasm increases. One indication of this is the steady increase of respiration throughout the period, which is generally attributed to an increase in the amount of active cytoplasm.

Much of the protein newly produced during cleavage is directly involved in the process of cell multiplication. One group of such proteins is the nuclear histones, which are needed for the chromosome replication in the same degree as additional quantities of DNA. In mid-cleavage of sea urchin embryos as much as 50 per cent of the newly synthesized protein is located in the nucleus.

The mRNA for these proteins is transcribed during cleavage and contrary to other mRNA’s, does not become masked, but is immediately used for translation into protein. This exception is probably due to the need for rapid synthesis of large quantities of nuclear histones. Some mRNA for nuclear histones is, however, present in the egg before fertilization.

Another protein synthesized during cleavage is tubulin, the constituent protein of microtubules—the fibers of the achromatic figures appearing during the mitotic divisions of cleavage cells. Tubulin is synthesized on messenger RNA already present in the egg. In the course of cleavage, tubulin is synthesized in increasing quantities, presumably as a result of progressive “unmasking” of the corresponding mRNA.

A third protein synthesized during cleavage is the enzyme ribonucleotide re­ductase, which in sea urchin embryos converts cytoplasmic ribonucleotides into deoxyribonucleotides, and thus provides a source of material for the replication of the chromosomal DNA.

The messenger RNA for ribonucleotide reductase is present in the unfertilized egg, but becomes active (is unmasked) after fertilization. A fourth protein necessary for chromosomal replication, the DNA polymerase, is already present in necessary quan­tities in the egg, and its quantity does not increase during early development.

If cleaving eggs are treated with puromycin, which inhibits RNA dependent protein synthesis, cleavage stops immediately, thus showing that protein synthesis is indispens­able for cleavage to take place. This is in marked contrast to cleavage being able to proceed in the presence of actinomycin D which effectively prevents the production of new RNA and particularly of new messenger RNA.

Possibly the most important of the proteins which have to be synthesized for cleavage to proceed are those which are used in the replication of the chromosomes – the nucleohistones, actually incorporated into the chromosome structure, and the ribonucleotide reductase, without which the cells are unable to use the supplies of RNA in the cytoplasm for replication of the nuclear DNA.

The synthesis of tubulin seems to be less important, as asters may be formed in the cytoplasm in the presence of puromycin, but do not lead to cell division. Presumably there is enough tubulin in the egg for aster formation, without new synthesis.

Patterns of Cleavage:

The way in which the egg is subdivided into the daughter blastomeres is usually very regular. The plane of the first division is, as a rule, vertical; it passes through the animal-vegetal axis of the egg. The plane of the second division is also vertical and passes through the animal-vegetal axis, but it is at right angles to the first plane of cleavage.

The result is that the first four blastomeres all lie side by side. The plane of the third division is at right angles of the first two planes and to the animal-vegetal axis of the egg. It is therefore horizontal or parallel to the equator of the egg. Of the eight blastomeres, four lie on top of the other four, the first four comprising the animal hemisphere of the embryo, the second the vegetal hemisphere.

If each of the blastomeres of the upper tier lie over the corresponding blastomeres of the lower tier, the pattern of the blastomeres is radially symmetrical. This is called the radial type of cleavage.

In many animals, however, the upper tier of blastomeres may be shifted with respect to the lower tier, and the radially symmetrical pattern becomes distorted in various degrees. The distortion may sometimes be due to individual variation, but there are certain groups of animals in which distortion always takes place and is the result of a specific structure of the egg.

In the annelids, molluscs, nemerteans, and some of the planarians (the Poly-cladida), all the blastomeres of the upper tier are shifted in the same direction in relation to the blastomeres of the lower tier, so that they come to lie not over the corresponding vegetal blastomeres, but over the junction between each two of the vegetal blastomeres.

This arrangement comes about not as a result of secondary shifting of the blastomeres, but because of oblique positions of the mitotic spindles, so that from the start the two daughter cells do not lie one above the other. The four spindles during the third cleavage are arranged in a sort of spiral. This type of cleavage is therefore called the spiral type of cleavage.

The turn of the spiral as seen from above may be in a clockwise direction or in a counterclockwise direction. In the first case the cleavage is called dextral; in the second case it is called sinistral. Since the cleavage planes are at right angles to the spindles, they also deviate from the horizontal position found in radial cleavage, and each cleavage plane is inclined at a certain angle.

The spiral arrangement of the mitotic spindles can be traced even in the first two divisions of the egg; the spindles are oblique and not vertical as in radial cleavage. However, the resulting shifts in the position of the blastomeres are not as obvious as after the third cleavage.

During the subsequent cleavages the spindles continue to be oblique, but the direction of spiraling changes in each subsequent division. Dextral spiraling alternates with sinistral, so that the spindle of each subsequent cleavage is approximately at right angles to the previous one.

Note that the type of cleavage of the egg as a whole, whether dextral or sinistral, depends on the direction of spiraling occurring during the third division of the egg.

Peculiarities of the cleavage pattern can also be introduced by differences in the size of the blastomeres. Of the four blastomeres in the four-cell stage of eggs having a spiral type of cleavage, one blastomere is often found to be larger than the other three.

This allows us to distinguish the individual blastomeres. The four first blastomeres are denoted by the letters A, B, C, D, the letters going in a clockwise direction (if the egg is viewed from the animal pole) and the largest blastomere being denoted by the letter D.

In some animals which otherwise have an approximately radial type of cleavage, two of the first four blastomeres may be larger than the other two, thus establishing a plane of bilateral symmetry in the developing embryo. Subsequent cleavages may make the bilateral arrangement of the blastomeres still more obvious (as in tunicates and in nematodes, although in a different way). The resulting type of cleavage is referred to as the bilateral type.

A very special type of cleavage showing bilateral symmetry is found in nematodes. The first division produces two unequal cells – a slightly larger cell designated as cell AB and a smaller cell P₁. The two cells divide next in mutually perpendicular planes, so that the blastomeres in the four-cell stage are placed in the form of a letter T.

The transverse shaft of the T is made up of blastomeres A and B (descendants of the cell AB), and the vertical shaft is made up of the offspring of blastomere P₁. The cells are designated as EMSt (abbreviation for endoderm, mesoderm, stomodeum—which shows the destiny of this cell) and as P₂. The “T” arrangement is, however, only temporary, the P₂ cell soon shifting toward the B cell.

The blastomeres are then arranged in a rhomboid figure. Next, the third division enhances the bilateral symmetry of the embryo, because the blastomeres A and B each divide into a right and left daughter cell, while the other two blastomeres produce a group of four cells lying one behind the other in the median plane. The blastomeres of this group are designated Mst, E, P₃, and C, respectively.

The cleavage in nematodes is also an example of determinate or mosaic cleavage in which definite blastomeres give rise to specific parts of the embryo. Thus, blastomeres A, B, and C give rise to the skin of the animal, blastomere E gives rise to the endoderm of the alimentary tract, blastomere MSt gives rise to the mesoderm and the stomodeum, and blastomere P₃ eventually produces the reproductive cells.

From the stage of eight cells a slight asymmetry is noticeable between the right and the left halves of the embryo.

The yolk, which is present in the egg at the beginning of cleavage in greater or lesser quantities, exerts a very far-reaching effect on the process of cleavage. Every mitosis involves movements of the cell components—the chromosomes, parts of the cytoplasm constituting the achromatic figure, the mitochondria, and the surface layer of the cell—the activity of which along the equator of the maternal cell leads to the eventual separation of the daughter cells.

During these movements, the yolk granules or platelets behave entirely passively and are passively distributed among the daughter blasto­meres. When the yolk granules or platelets become very abundant, they tend to retard and even to inhibit the process of cleavage.

As a result, the blastomeres which are richer in yolk tend to divide at a slower rate and consequently remain larger than those which have less yolk. The yolk in the uncleaved egg is more concentrated toward the vegetal pole of the egg. It is therefore at the vegetal pole of the egg that cleavage is most retarded by the presence of yolk, and where the blastomeres are of the largest size.

A good example of the effect of the yolk on cleavage is provided by the frog’s egg. The yolk’s influence may be detected even during the first division of the fertilized egg. During the anaphase of the mitotic division, a furrow appears on the surface of the egg which is to separate the two daughter blastomeres from each other.

This furrow, however, does not appear simultaneously all around the circumference of the egg, but at first only at the animal pole of the egg, where there is less yolk. (It has been indicated that the first cleavage plane is vertical and therefore passes through the animal and vegetal poles of the egg.)

Only gradually is the cleavage furrow prolonged along the meridians of the egg, until, cutting through the mass of yolk-laden cytoplasm, it eventually reaches the vegetal pole and thus completes the separation of the first two blastomeres.

The same process is repeated during the second cleavage. During the third cleavage, when the plane of separation of the daughter blastomeres is horizontal, the furrow appears simultaneously over the whole circumference of the egg, for it meets everywhere with an equal resistance from yolk.

A further accumulation of the yolk at the vegetal pole of the egg causes still greater delay in the cell fission at this pole, so that the cleavage becomes inhibited more and more. This can be clearly traced in a series of various ganoid fishes, whose eggs possess an increasing amount of yolk.

In Acipenser the cleavage is complete, as in the amphibians, but the difference between the micromeres of the animal hemisphere and the macro-meres of the vegetal hemisphere is much greater than in amphibians. In Amia cleavage starts at the pole, and the cleavage furrows reach the vegetal pole, but they are so retarded that subsequent divisions begin at the animal pole before the preceding furrows cut through the yolk at the vegetal pole.

In Lepidosteus the cleavage starts at the animal pole as in Amia, but the cleavage furrows never reach the vegetal pole, so that the vegetal hemisphere of the egg remains un-cleaved, resulting in what is called incomplete cleavage. The type of cleavage during which the whole of the egg becomes subdivided into blastomeres is called holoblastic (complete) cleavage.

The type of cleavage in which only a part of the egg is subdivided into blastomeres is called meroblastic (incomplete) cleavage. As the result of meroblastic cleavage the egg is divided into a number of separate blastomeres, and a residue, which is a continuous mass of cytoplasm, usually with some nuclei scattered in it.

Sometimes the terms holoblastic and meroblastic are applied also to the eggs having a particular type of cleavage; thus, one finds in the literature the term “holoblastic eggs,” meaning eggs having holoblastic cleavage, and also the term “meroblastic eggs” for eggs having meroblastic cleavage.

In eggs in which the yolk is segregated from the active cytoplasm (elasmobranchs, bony fishes, birds, and reptiles), the cleavage, right from the start, is distinctly recogniza­ble as meroblastic or incomplete. At first, all the cleavage planes are vertical, and all the blastomeres lie in one plane only.

The cleavage furrows separate the daughter blastomeres from each other but not from the yolk, so that the central blastomeres are continuous with the yolk at their lower ends, and the blastomeres lying on the circumference are, in addition, continuous with the un-cleaved cytoplasm at their outer edges. As the nuclei at the edge divide, more and more cells become cut off to join the ones lying in the center, but the new blastomeres are also in continuity with the un-cleaved yolk underneath.

In a later stage of cleavage, the blastomeres of the central area become separated from the underlying yolk in one of two ways – either slits may appear beneath the nucleated parts of the cells, or else the cell divisions may occur with horizontal (tangen­tial) planes of fission. In the latter case one of the daughter cells, the upper one, becomes completely separated from its neighbors, while the lower blastomere retains the connec­tion with the yolk mass.

The marginal cells, which remain continuous with one another around their outer edges, are also continuous with the mass of yolk and hence with the lower cells resulting from tangential divisions. All these blasto­meres eventually lose even those furrows which partially separated them from one another and fuse into a continuous syncytium with numerous nuclei but no indication of individual cells.

Nuclei of Cleavage Cells:

In his “germ plasm theory,” A. Weismann presented a hypothesis to explain both heredity and the ontogenetic development (Metazoa) of organisms. According to Weismann (1904), every distinct part of an organism (animal or plant) is represented in the sex cell by a separate particle – a determinant.

Thus, the sum total of determinants would represent the parts of the adult organism with all their peculiarities. The complete set of determinants is supposedly handed down from generation to generation, which would account for hereditary transmission of characters.

The determinants, according to Weismann, are localized in the chromosomes of the nucleus, just as the genes of modern genetics are. However, there is a difference – the genes are not supposed to represent parts of the organism but rather properties which may sometimes be discernible in all the parts of the body.

During the cleavage of the egg, the various determinants, according to Weismann, become segregated into different cells. The blastomeres would receive only some of the determinants, namely, those which correspond to the fate of each blastomere.

The successive segregation of the determinants would eventually result in each cell’s having determinants of only one kind, and then nothing would be left to the cell but to differentiate in a specific way in accordance with the determinants present. Only the cells having the sex cells among their descendants, Weismann held, would preserve the complete set of determinants, since these would be necessary for directing the de­velopment of the next generation.

Even though Weismann’s conception of the properties of determinants is not tenable from the genetic viewpoint, it is still important to know whether the difference in the fate of the blastomeres and parts of the embryo may be attributed to differences in the nuclei of the cleavage cells.

Our knowledge of the development of complete embryos from one of the two daughter blastomeres of the egg (as a result of either separating the first two blastomeres or killing one of the first two blastomeres) contradicts Weismann’s hypothesis about the segregation of determinants during cleavage.

The evidence be­comes still more conclusive because it has been found that not only are the first two blastomeres capable of developing into complete embryos, but the blastomeres in later cleavage stages sometimes possess the same ability. In the case of sea urchins, one of the first four blastomeres, and even occasionally one of the first eight, may develop into a whole embryo with all the normal parts but reduced in size.

The method of isolating blastomeres, however, does not permit one to test the properties of nuclei of later generations of cells. In the four-cell stage, the quantity of cytoplasm contained in one blastomere may already be too small for development to take place in an approximately normal fashion. This is true in increasing degree as cleavage proceeds and the individual blastomeres become smaller and smaller.

To further this investigation, a different method has been devised. A most elegant experiment in this field was carried out by Spemann (1928). Spemann constricted fertilized eggs of the newt Triturus (Triton) into two halves with a fine hair, just as they were about to begin to cleave.

The constriction was not carried out completely, so that the two halves were still connected to each other by a narrow bridge of cytoplasm. The nucleus of the fertilized egg lay in one half, and the other half consisted of cytoplasm only. When the egg nucleus began to divide, the cleavage was at first restricted to that half of the egg which contained the nucleus.

This half divided into two, four, eight cells, and so on, while the non-nucleated half remained un-cleaved. At about the stage of 16 blastomeres, one of the daughter nuclei, now much smaller than at the beginning of cleavage, passed through the cytoplasmic bridge into the half of the egg which had hitherto no nucleus.

Forthwith this half also began to cleave. After both halves of the egg were thus supplied with nuclei, Spemann drew the hair loop tighter and completely separated the two halves of the egg from each other. They were then allowed to develop into embryos. In a number of cases two completely normal embryos developed from the two halves of the egg.

Of the two embryos, each started by having one half of the egg cytoplasm, but as to the nucleus they were in very different situations. While one of the embryos possessed 15/16 or even 31/32 of all the nuclear material of the egg, the other received only 1/16 or 1/32 of the nuclear material. The experiment proves conclusively that even in the 32-cell stage every nucleus has a complete set of hereditary factors necessary for the achievement of normal development.

All the nuclei in this stage are completely equiva­lent to one another and to the nucleus of the fertilized egg. The hypothesis of an unequal division of the hereditary substance of the nucleus, of the segregation of determinants or genes to the different cleavage cells, is thus disproved. It is now assumed that every cell of the metazoan body has a complete set of nuclear factors necessary for development (a complete set of genes, in the terminology of modern genetics).

The experiments on the delayed nuclear supply to one half of an amphibian egg have been corroborated by experiments on the eggs of other animals. It will be useful to relate a corresponding experiment carried out on a very different kind of animal, the dragonfly Platycnemis pennipes.

In the dragonfly, cleavage is incom­plete, and only the nucleus divides at first, the cytoplasm remaining un-cleaved. The egg is elongated, and after the first division the daughter nuclei move, one into the anterior half and the other into the posterior half of the egg. When eight nuclei are available, they are spaced along the length of the embryo.

By further divisions nuclei are provided for all cells in the respective regions. In the stage when two nuclei are present, either of them may be destroyed by a short exposure to a narrow beam of ultraviolet light, which does not damage the cytoplasm to any great extent.

The remaining nucleus continues to divide, and its daughter nuclei are distributed to all parts of the egg instead of supplying only one half of it. Completely normal embryos develop, no matter which of the two nuclei is allowed to survive. The two nuclei prove to be completely equivalent for development, although normally they would have supplied different parts of the embryo.

The methods used in the preceding experiments for testing the properties of the cleavage nuclei are of necessity confined to the earlier stages of cleavage. At present, a more universal method is available which allows the investigation to be extended to nuclei of cells of much more advanced embryos, and possibly it may ultimately be applied even to fully differentiated cells of an adult organism.

This is the method used for transplantation of nuclei. The transplantation of nuclei from one cell to another was first carried out successfully on Amoeba, and the method was then applied to test the properties of nuclei in developing frog embryos. The method, as applied to the frog embryo, consists essentially in taking the nucleus of any cell from a developing embryo and injecting it into an enucleated un-cleaved egg.

The egg receiving the nucleus must be specially prepared. The ripe eggs are removed from the oviducts and activated by pricking with a glass needle. The egg nucleus then approaches the surface of the cytoplasm in preparation for the second maturation division and is removed by a second prick with a glass needle at the exact spot where the nucleus is located.

Next, a cell of an advanced embryo is separated from its neighbors and sucked into the tube of an injection pipette. The diameter of the pipette is smaller than that of the cell and as a result the surface of the cell is broken. The contents of the pipette, consisting of the nucleus and the debris of the cytoplasm, are injected deep into the enucleated egg.

When the pipette is withdrawn, the egg cytoplasm tends to escape through the hole in the egg membranes, forming an extraovate protrusion. This must be cut off by a pair of glass needles to prevent further loss of egg substance.

As a result of these procedures, up to 80 per cent of the eggs operated on start cleaving and producing numerous cells, the nuclei of which are derived from the injected nucleus and the cytoplasm of which is from the enucleated egg (the small amount of cytoplasm injected with the nucleus, comprising less than 1/40,000 of the volume of the egg cytoplasm may be ignored). Not all eggs which start cleaving develop normally later, but at least a small proportion do and may proceed through all the stages of embryonic and postembryonic development up to metamor­phosis.

In the late blastula, the stage used for some of these nuclear transplanta­tions, there are 8000 to 16,000 cells, which means that about 13 to 14 divisions (or generations) of the original nucleus of the fertilized egg had been performed without diminishing the power of the nucleus to support every type of differentiation provided for by the specific genotype. Nuclei of cells of an early gastrula show the same properties.

In further experiments the potentialities of nuclei of cells, which are even more advanced in the process of differentiation, were tested by transplanting them into enucleated eggs. Normal tadpoles developing up to and through metamorphosis were obtained by using nuclei from the neural plate of a frog embryo or nuclei from already ciliated cells of the alimentary tract of a swimming tadpole.

Although the cells of the neural plate were already well on the way to becoming cells of the nervous system, and the cells of the gut were already functionally differentiated, their nuclei were still capable of providing the necessary genetic information for the differentiation of all the various tissues and cell types of an adult frog. Fairly normal embryos were also produced by implanting into enucleated eggs the nuclei from a frog adenocarcinoma.

Finally, a modification of the original methods has made it possible to test the potentialties of nuclei of adult animals by transplanting them into enucleated eggs. When nuclei are taken directly from adult tissues and transplanted into eggs, they are not able to support development.

If adult cells are cultured in vitro, however, where they lose part of the properties of differentiated cells and start reproducing mitotically, and if their nuclei are then transplanted into eggs, development may be initiated in a fair proportion of cases.

Even so, the embryos develop abnormally, producing blastulae with mostly partial cleavage. If nuclei of cells from the more healthy parts of the partial embryos are then removed and transplanted into eggs, the development of such second generation embryos proceeds much better; swimming larvae and occasionally even larvae which become metamorphosed into froglets develop.

The tissues used success­fully as sources of nuclei are kidney, lung, and skin of adult frogs. It is noteworthy that no differences in the development of tadpoles could be noted when the nuclei were taken from these three different tissues.

It was noted that with progression of development of the cells from which nuclei were taken the proportion of experiments leading to completely normal tadpoles became increasingly reduced, and more and more operated eggs were arrested in their development in various early stages.

On careful investigation it was found that the chromosomes in the nuclei of arrested embryos showed various defects, such as aneuploidy (chromosomes missing) or defects within chromosomes (deletions, translo­cations).

These defects are due to the inability of the chromosomes in nuclei taken from advanced embryos and adult tissues to adapt themselves to the rapid reproductive rhythm of early development. The duplication of the interphase chromosomes is too slow, and many of them do not complete duplication by the time cell division sets in.

The result is, of course, severe damage to and incompleteness of the chromosome set. Whatever the explanation for the defective development, it remains that at least a proportion of nuclei from cells well on the way to differentiation and from those that have become malignant retain their full potentialities for controlling and directing normal development.

Nuclei of differentiating cells are in every respect similar to the nuclei of fertilized eggs. In experiments with frogs, nuclei transplanted into the egg cytoplasm undergo a change which makes them resemble early embryonic nuclei.

The nuclei of newly fertilized eggs, although small in proportion to the egg cytoplasm, are much larger than the nuclei at later stages. Accordingly, transplanted nuclei increase up to 30-fold in volume during the first 40 minutes after transplantation. The functioning of the nuclei also changes drastically.

Nuclei of ad­vanced embryos do not divide rapidly, and accordingly the synthesis of DNA, necessary for the replication of chromosomes, is slow. On the other hand, they synthesize large quantities of ribosomal RNA, and, as this kind of RNA is produced in the nucleolus, they have prominent nucleoli.

Nuclei of cleavage cells synthesize DNA rapidly but do not synthesize any ribosomal RNA and therefore do not show any nucleoli. Transplanted nuclei cease to synthesize ribosomal RNA, and their nucleoli disappear. Instead, they start to synthesize DNA rapidly as do normal cleavage nuclei.

Morula and Blastula of Cleavages:

The blastomeres in the early cleavage stages tend to assume a spherical shape like that of the egg before cleavage. Their mutual pressure flattens the surfaces of the blastomeres in contact with one another, but the free surfaces of each blastomere remain spherical, unless these outer surfaces are also compressed by the vitelline membrane. The whole embryo acquires, in this stage, a characteristic appearance resembling a mulberry. Because of this superficial similarity, the embryo in this stage is called a morula (Latin for mulberry).

The arrangement of the blastomeres in the morula stage may vary in the different groups of the animal kingdom. In coelenterates it is often a massive structure, with blastomeres filling all the space that had been occupied by the un-cleaved egg. Some of the blastomeres then lie externally and others in the interior. (Some embryologists apply the name morula to this type of embryo only.)

More often, as the egg undergoes cleavage, the blastomeres become arranged in one layer, so that all the blastomeres participate in the external surface of the embryo. In this case a cavity soon appears which at first may be represented just by narrow crevices between the blastomeres, but which gradually increases as the cleavage goes on. This cavity is called the blastocoele.

As cleavage proceeds, the adhesion of the blastomeres to one another increases, and they arrange themselves into a true epithelium. In cases in which a cavity has been forming in the interior of the embryo, the epithelial layer completely encloses this cavity, and the embryo becomes a hollow sphere, the walls of which consist of an epithelial layer of cells. Such an embryo is called a blastula. The layer of cells is called the blastoderm, and the cavity is the blastocoele.

Right from the beginning of cleavage the blastomeres become progressively joined closer together by several types of intercellular junctions. The first to be formed are the “tight” junctions forming at the outer edges of the adjoining blastomeres, sometimes already in the two-blastomere stage. At the blastula stage these junctions seal off the interior of the embryo from the outside.

In the sea urchin blastula these junctions take the form of “septate” junctions, which appear as a series of bars connecting the membranes of adjoining cells. “Gap” junctions which provide a means of communication between cells of the embryo are absent in the earliest stages, but develop in the morula stage and are widespread in the blastula stage.

In oligolecithal eggs with complete cleavage (echinoderms, Amphioxus), the blas­tomeres at the end of cleavage are not of exactly equal size, the blastomeres near the vegetal pole being slightly larger than those on the animal pole. When the blastula is formed, the cells arrange themselves into a simple columnar epithelium enclosing the blastocoele.

Because the vegetal cells are larger than the animal cells, the blastoderm is not of an equal thickness throughout; at the vegetal pole the epithelium is thicker, and at the animal pole it is thinner. Thus, the polarity of the egg persists in the polarity of the blastula.

Animals with a larger amount of yolk, such as the frog, show a difference in the size of the cells of the blastula that may be very considerable, and the blastula still further departs from the simple form of a hollow sphere. The cells here are also arranged in a layer surrounding the cavity in the interior, but the layer is of very uneven thickness.

The layer of cells at the vegetal pole is very much thicker than at the animal pole, and the blastocoele is consequently distinctly eccentric, nearer to the animal pole of the embryo. Furthermore, the blastoderm is no longer a simple columnar epithelium but is two or more cells thick.

The cells in the interior are rather loosely connected to one another, but at the external surface of the blastula the cells adhere to one another very firmly, because of the presence of tight cell junctions joining the surface of adjoining cells in a narrow zone just underneath the surface of the blastoderm.

A process corresponding to blastula formation occurs also in animals whose eggs have incomplete cleavage. In a bony fish or a shark the early blastomeres tend to round themselves off, showing that they are only loosely bound together. Later, the blasto­meres adhere to one another more firmly and thus become converted into an epithelium.

Again, as in amphibians, the superficial cells are firmly joined to one another, forming a continuous “covering layer,” while the cells in the interior may remain loosely connected until a later stage. The epithelium, however, cannot have the form of a sphere.

Since the cleavage is restricted to the cap of cytoplasm on the animal pole of the egg, the blastoderm is developed only in the same polar region. The blastoderm therefore assumes the shape of a disc lying on the animal pole. The disc, which is called the blastodisc, is more or less convex and encloses, between itself and the un-cleaved residue of the egg, a cavity representing the blastocoele.

In centrolecithal eggs having a superficial cleavage (insects), there is no cavity comparable to the blastocoele. Nevertheless, the formation of the epithelium on the surface of the embryo, after the nuclei have migrated to the exterior, can be compared to the formation of the blastula.

The layer of cells thus formed on the surface of the embryo is the blastoderm. Instead of surrounding a cavity, the blastoderm envelops the mass of un-cleaved yolk. We may also compare this stage to an embryo whose blastocoele has been filled with yolk.

Up to the blastula stage, the developing embryo preserves the same general shape as the un-cleaved egg. So far, the results achieved are the subdivision of the single cell into a multiplicity of cells and the formation of the blastocoele. In addition, the sub­stances contained in the egg remain basically in the same position as before.

The yolk remains near the vegetal pole. In pigmented eggs, such as those of amphibians, the pigment remains as before, more or less restricted to the upper hemisphere of the embryo. Only a slight intermingling of cytoplasm seems to be produced by the cleavage furrows cutting through the substance of the egg.

We have pointed out that during cleavage qualitative changes in the chemical composition of the developing embryo are very limited. Few new substances, either chemically defined or microscopically detectable, have been found to appear during cleavage. It is conceivable, however, that the substances present in the egg may be redistributed in some way during cleavage and that such a redistribution may be essentially important for further development.

In this connection we will first examine whether the numerous nuclei produced during the mitotic divisions of the egg are all alike in their properties, or whether any differences may be discovered among them.