Waddington1956 4

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Waddington CH. Principles of Embryology (1956) The MacMillan Co., New York

   Principles of Embryology (1956): Part 1 - 1 The Science of Embryology | 2 The Gametes | 3 Fertilisation | 4 Cleavage | 5 The Echinoderms | 6 Spirally Cleaving Eggs | 7 The Ascidians and Amphioxus | 8 The Insects | 9 The Vertebrates: The Amphibia and Birds | 10 The Epigenetics of the Embryonic Axis | 11 Embryo Formation in Other Groups of Vertebrates | 12 Organ Development in Vertebrates | 13 Growth | 14 Regeneration | 15 The Role of Genes in the Epigenetic System | 16 The Activation of Genes by the Cytoplasm | 17 The Synthesis of New Substances | 18 Plasmagenes | 19 The Differentiating System | 20 Individuation - The Formation of Pattern and Shape | References
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-Chapter IV Cleavage=

1. General features

In all eggs, fertilisation is followed by a period in which the egg divides into a number of cells. This is known as the period of cleavage. It may be said to last until some other process, besides mere subdivision, begins to become important; the process in question is usually a shifting of regions of the egg relative to one another—a series of movements which, as we shall see, will eventually bring about gastrulation. By the time these movements begin the egg has been extensively cut up into smaller cells, and from this time onwards the zygote is commonly referred to as an ‘embryo’ instead of, loosely, as an ‘egg’. As we saw in Chapter I the cleavage cells at the beginning of gastrulation are arranged as a ‘blastula’, which in its most typical form is a hollow sphere. We shall find, however, that the course taken by the cleavages in many groups is such that the blastula is considerably modified from this pattern.

The main physiological function of cleavage is to redress the balance between the size of the nucleus and the volume of cytoplasm with which it is associated. Egg-cells are always very large, as cells go. During the growth period of the oocyte, the nucleus is also large, being distended to form the germinal vesicle. But after fertilisation, the new zygote nucleus is of about normal size for the species in question, and it finds itself in a cell which is far larger than normal growing cells (although some fully differentiated cells are again very large). During cleavage, the balance is restored. This involves not only a reduction in cell-size by subdivision, but also the formation of new nuclear material to build up the increased number of nuclei. The two most important classes of substance required for these nuclei are the proteins and desoxyribose nucleic acid which together make up the chromosomes. Very little is known of the source of origin of the chromosomal proteins, which cannot easily be isolated from the other proteins of the egg. Technical methods are available for studying the nucleic acids, including the ribose nucleic acid (RNA).which is characteristically present in the cytoplasm as well as the desoxyribose (DNA) compound found in the chromosomes. It was at one time suggested (by Brachet) that in many invertebrates the DNA was formed by conversion of the cytoplasmic RNA (a ‘partial synthesis’) but this now seems to be unlikely. It has in fact recently been argued (Zeuthen 1951, Hoff-Jorgensen 1954) that in many forms no net synthesis of DNA takes place during the early part of cleavage, since the cytoplasm of the egg contains stores of this substance which are sufficient to provide for many» cleavage nuclei—perhaps a few million in the chick, and a few thousand in the frog, though only about sixteen in the sea-urchin. While this DNA is being incorporated into the nuclei, it is in a state of metabolic activity, since radio-active phosphate is rapidly taken into it (e.g. Villee and Villee 1952); probably also changes are going on in its specificity, converting it into material capable of acting as genetic determinants, but little is known about this.

In the readjustment of the nuclear-cytoplasmic ratio, subdivision of the active protoplasm is of much more importance than cutting up of the inactive yolk, which probably makes no chemical demands on the nucleus. The progress of cleavage is accordingly always profoundly modified by the presence of appreciable quantities of yolk. We find, for instance, that in yolky eggs, the fertilised nucleus is displaced from the centre of the egg towards the less yolky end. Moreover, the cleavages begin earlier and go on faster in the less yolky parts. And very often the cleavage spindles are orientated so as to lie with their axes in the direction of the longest stretch of non-yolky cytoplasm available in the cell—but we shall see that this rule (sometimes known as Balfour’s rule) is not the only factor at work in controlling the spindle directions, since these may be definitely orientated even in eggs where there is too little yolk to make Balfour’s mechanism effective.

The result of these factors is that in eggs with a fair amount of yolk, the cleavages produce more and smaller cells in the less-yolky animal region than in the more yolky vegetative end. Where the store of yolk is very large, the most heavily laden region may not be divided at all during cleavage; in fact, in extreme cases such as reptile and bird eggs, cleavage is confined to a small superficial area near the animal pole, where alone there is any appreciable quantity of cytoplasm (Fig. 4.1).

We may therefore classify cleavage types as follows:

Total cleavage. Whole egg divides.

(i) Equal. In eggs with little yolk.

(ii) Unequal. In eggs with rather more yolk. Partial cleavage. Part of the egg remains undivided; in eggs with rather large stores of yolk. Superficial cleavage. Cleavage only in a small area of the egg; in extremely yolky eggs, CLEAVAGE 59

Total cleavage

Total cleavage is the rule in the small, non-yolky eggs of most marine invertebrates. In the eggs of most species of echinoderms, molluscs, coelenterates, worms, etc., only rather little yolk can be distinguished. The first cleavages in such eggs usually cut them into cells of roughly equal size; such cleavage cells are often known as blastomeres. In most cases the equality between them is not very exact, and it usually does not last through many cleavage divisions. Indeed, the most interesting aspect of the cleavage of these eggs is the fact that there are characteristic patterns of large and small cells into which the eggs of a particular group become divided. In such patterns, there is often no obvious relation between the size of the cell and the yolk content. The cleavage pattern must be determined in some other way (cf. p. 67).

File:Waddington1956 fig4.1.jpg

Figure 4.1.

A. Lateral view of very unequal cleavage in the yolky egg of a sturgeon.

B. Diagrammatic section of a late cleavage stage in the chick. The blasto derm is beginning to delaminate into two layers, the hypoblast (endoderm) below and the epiblast (ectoderm and mesoderm) above. S.B.C. subblastodermic cavity.

Examples of the main cleavage patterns are described in more detail in the chapters dealing with the different groups. Here we shall merely list them, for reference.

  1. Radially symmetrical. Best scen in echinoderms. In these the first two cleavages are vertical, the third horizontal, but a more complicated pattern begins to appear at the fourth division, in which the cleavage plane is vertical in the animal half and horizontal in the vegetative half, where it cuts off a lower ring of quite small cells, known as sicromeres.
  2. Bilaterally symmetrical. In a great many eggs, the first two cleavages are vertical, passing through the animal-vegetative axis, and cut off four cells which are not equal, but arranged with a bilateral symmetry. This is true, for instance, in some coelenterates (e.g. the ctenophore Beroé); in ascidians and Amphioxus; and in most vertebrates where the accumulation of yolk is not so large as to disturb the picture completely.
  3. Spirally symmetrical. There is a large group of invertebrates (nemerteans, annelids, molluscs) in which the first two cleavages are not quite vertical, but slightly inclined, so that when looked at from the animal pole, the first four cells are arranged with a slight spiral twist. The third cleavage plane is more or less horizontal, and cuts off a ring of micromeres at the animal end; and these again do not lic immediately above the lower ring of four cells, but are twisted out of place. The subsequent course of cleavage in these eggs has been studied in great detail, particularly by a group of American authors at the beginning of the century (Conklin and E. B. Wilson are perhaps the best known of these). It was shown that the various cells formed after the first five or six cleavages regularly develop into definite parts of the embryo, so that the cleavage pattern is very intimately involved in the developmental processes; we shall see (p. 62) that this is by no means usual in other types of cleavage (Fig. 4.2). In many animals the simple spiral pattern is modified by a rather remarkable process; just before the first division, the egg pushes out a large pseudopodium-like excrescence, which is called the ‘polar lobe’ since it forms near the vegetative pole (Fig, 6.4, p. 100). The cleavage runs so that the whole of this lobe becomes incorporated into one of the two daughtercells. And the process is repeated through several of the later divisions. It appears to be a mechanism for temporarily putting certain material on one side. The cell into which this material eventually comes is the one from which the mesoderm is developed, and thus one of the most important for the future development. Moreover, it has recently been possible to cause an egg with a polar lobe to cleave in such a way that the polar material is divided among the first two cells; it was found that a double embryo was formed (p. 99). This demonstrates the essential role which this material plays in development, and enables us to understand why a special mechanism has been evolved to keep it intact while it is being sorted out into the final mesoderm-forming cell. The result also shows that the spirally cleaving egg is not a true mosaic of parts whose fates are irrevocably fixed, since in the formation of such double embryos a good deal of regulation must have been involved.
  4. Irregular cleavage. In some coelenterate eggs, the cleavage pattern is quite irregular; indeed the blastomeres tend to fall apart and are only held loosely together by the jelly in which they are embedded. Perhaps one can also include, under the heading of irregular cleavage, the phenomena found in insects, in which the cleavage consists only in the separation of daughter nuclei within the undivided mass of the yolky egg (see p- 119). Unequal, partial and superficial cleavage

The most important examples of unequal cleavages are found in vertebrates, which present a complete series of types, from those in which there is only a minor difference between the animal and vegetative cells, to those in which cleavage is confined to a relatively tiny area at the animal pole. The frog or newt are good examples of slightly unequal cleavages, some of the cartilaginous fishes are the best intermediate types, while the reptiles, teleosts and birds are the classical ‘superficial’ types.

File:Waddington1956 fig4.2.jpg

Figure 4.2.

Spiral cleavage (dextral) seen from the animal pole. At the 4-cell stage, the right-or left-handedness of the cleavage can already be recognised by the direction of the cross-furrow at the animal pole. At the third cleavage, the macromeres A, B, C, D give off micromeres 1a, 1b, 1c, 1d, the cleavage spindles being tilted in the direction indicated by the arrows. At the next cleavage, the second quartet of micromeres, 2a, 2b, etc., are formed, with the spindles tilted in the other direction; and 14 divides into 1at and 1a2, tb into 1br and 1b2, etc. This system of cleavage continues until four quartets of micromeres have been formed; but the divisions of the micromeres and macromeres are not always synchronous, cleavage of 1a into 1a1, 142 being sometimes delayed till after the formation of the second quartet, and so on. 62 PRINCIPLES OF EMBRYOLOGY

In the more primitive, nearly equal, members of this series (such as Amphibia) the pattern seems to be one of bilateral symmetry, the first two cleavages being vertical, and forming four cells of which one pair is slightly larger than the other. The third cleavage is horizontal, but lies above the equator, so that the less yolky animal cells are smaller than the vegetative ones. This difference is greatly exaggerated in the more yolky eggs, such as those of sturgeons. But in the most yolky eggs there is a sharply different type of cleavage. The small animal cells do not gradually shade off into larger and larger blastomeres; instead, the cleavage occurs only in the animal cytoplasmic region, in which all the cleavage cells are of similar size, while the main mass of yolk remains completely undivided. There is thus a rather sharp boundary between the cellular and non-cellular parts of the egg. (There may be a few nuclei scattered in the yolk just near the border of the cellular region, these being derived from supernumerary sperm which enter the egg outside the sphere of influence of the primary fertilising sperm.) This is the condition in reptiles and birds.

In a very rough way, the series from non-yolky, totally cleaving eggs ~ up to very yolky, superficially cleaving types parallels the evolutionary series of the vertebrates. But the parallel breaks down entirely when we come to the mammals. Since it is nurtured in the uterus, the mammal egg has no need of large stores of yolk, and in fact is not provided with them. And in the absence of yolk, the cleavage is total and more or less equal, though with no very well-defined symmetry pattern.

2. The pattern of cleavage and the pattern of the embryo

Since the cleavages frequently follow a definite and orderly pattern, it is perhaps natural to expect that this will be directly related to the pattern of the embryo which eventually develops. Many of the earliest studies on the physiology of development aimed at discovering whether this is so or not. It turns out that there is no simple, single answer which applies to all animals; in fact this is a question to which we shall have to return several times in the later discussions of the development of different groups. Even the very earliest experiments, at the end of the last century, showed that the subject was complicated. Driesch, in 1891, separated the first two blastomeres of an echinoderm egg, and found that each gave rise to a complete embryo, not to only half an embryo as would be expected if there is a direct relation between cleavage pattern and embryo. He concluded that any part of the egg, in the early cleavage stages, is capable of forming a whole animal; and he spoke of such eggs as ‘regulation’ eggs, all of whose parts are ‘equi-potential’ and capable of regulating so as to replace any part that might be removed. The critical reader will notice that Driesch drew conclusions about the behaviour of all parts from experiments in which he had actually only succeeded in isolating certain parts; thus he always cut the egg parallel to the first cleavage planes, which are vertical, and he was not justified in assuming that if he had cut it along a horizontal plane, those halves would also regulate completely. As we shall see later, in fact they do not do so; and it is probable that no egg is actually a completely regulation egg in Driesch’s sense.

Very soon after Driesch’s discovery, similar experiments on other eggs turned out in exactly the opposite way. Roux, for example, found that when one of the first blastomeres of a frog’s egg is killed, the other develops into half an embryo; and the same thing occurred in ascidians and many of the spirally cleaving eggs. Embryologists began to speak of ‘mosaic’ eggs, contrasting these with the regulation type, and supposing that they contained a mosaic of different cytoplasmic regions each of which irrevocably developed into some specific part of the embryo. But in this concept again they were going beyond their actual facts. The experimental results showed that when certain eggs were injured, the remaining parts did not regulate so as to compensate for the loss; but this does not necessarily imply that all regulation is impossible in such eggs. We shall see later that this is not only a logical non sequitur, but is controverted by the facts which have become known more recently. In fact, there seems to be no more a completely mosaic egg than a completely regulation one. All eggs, we shall find, partake of both characters; all have some definiteness of localisation of parts with specific properties; and all have some capacity for adjusting themselves to injuries. It is true that in some eggs the localisation is the more striking phenomenon, in others the regulation; and many eggs can still be looked on as tending to one or other extreme; but the differences are not absolute, and the two pure types do not exist.

3. Differentiation without cleavage

In the early years of the century F. R. Lillie discovered that parthenogenetically activated eggs of the polycheate Chaetopterus may sometimes achieve a considerable degree of differentiation although remaining in an undivided state. In these eggs the cleavages are, however, not totally suppressed. Pasteels (1934) and Brachet (1937), ‘who have re-studied the material more recently, point out that the activated egg becomes lobulated in a manner which closely simulates the normal cleavage pattern, and that there are cycles of activity of the nucleus, involving the appearance of asters, usually monocentric but occasionally leading to true mitoses. Although the lobulations eventually disappear, so that the egg regains its spherical shape, their occurrence is important evidence that autonomous cytoplasmic (probably cortical) changes play a part in the normal cleavage process (see also Lehmann 1948a).

The ‘differentiation’ performed by these eggs is also by no means complete. The most they accomplish is the separation into different regions of various types of cytoplasm, together with a relative movement of the outermost clear cytoplasm over the vegetative material which somewhat recalls the normal process of gastrulation, and finally a differentiation of cilia; but there is no true organogenesis, such as the formation of a gut or apical tuft. Nevertheless, the evidence that even this segregation of ooplasmic components can proceed as far as it does when the egg is not divided up into cells, indicates the importance of processes which go on within the body as a whole. It becomes clear that it is dangerous to attribute too much importance to the cell as the basic unit on which everything depends, and that theories such as that of Weiss (p. 413) which attempt to explain development in terms of the properties of cell membranes can be at best a part of the truth.

4. Cleavage without nuclei

It has been mentioned in the last chapter that parthenogenetically activated eggs or egg fragments may undergo fairly regular cleavages even in the absence of nuclei. Cases of this have been described by Harvey (1936, 1940) in echinoderms, by Gross (1936) in Artemia, and Fankhauser (1934) and Briggs, Green and King (1951) in Amphibia. Some authors question whether the phenomenon is truly comparable to cleavage, and suggest that it is more like the disorganised ‘bubbling’ that some cells undergo at the time of division, but in the best cases described in the Amphibia something very like a normal blastula is produced, and it seems unduly sceptical to deny the process the name of cleavage (Fig. 4.3).

5. The mechanism of cleavage

Cleaving eggs, particularly those of marine invertebrates, have frequently been used to study the general problems of cell division. This is an enormous subject, involving the behaviour of the chromosomes, of the achromatic apparatus (spindle, centrosomes, asters, etc.) and of the body of the cell which becomes divided in two. A full discussion of all aspects of it would lead us too far into the fields of cytology and cell physiology and we shall confine ourselves here to those aspects which are particularly important in relation to the problems of embryonic development. We shall therefore pay little attention to the subject of chromosome movements or the details of the behaviour of the achromatic apparatus (for which consult textbooks on cytology, such as Darlington 1939, White 1950, 1954, Schrader 1944, Hughes 1952). From the point of view of the embryologist, the important subjects to discuss are the determination of the pattern of cleavage and the mechanism by which the body of the cell becomes divided into two parts.

File:Waddington1956 fig4.3.jpg

Figure 4.3.

Section through the best cleaved portion ofa blastula, which was developed from an enucleated egg of the frog Rana pipiens, inseminated by a sperm of R. catesbiana whose nucleus had been inactivated by U.V. irradiation. The cells are outlined by pigment granules and the dark spots resembling nuclei are also accumulations of pigment. There is some chromatin in most cells in region a, in a few cells in region b, but none in the cells of region c. (From Briggs, Green and King 1951.)

The series of events by which a cell is cleaved in two is normally initiated by the nuclear division, during which the chromosomes become separated into two daughter groups. That there is a causal relation between the two processes is shown by the general concordance in their timing: nuclear division is usually followed immediately by cell division. The primacy of the former is shown by the fact that when, in abnormal cells or under experimental conditions, the usual sequence is disturbed, - it is the cleavage of the cell rather than the division of the nucleus which is most easily changed from its normal course. Thus it is not uncommon for nuclear divisions to occur without any following cleavage of the cell body, but it is very rare for the cell to cleave in a way which is not dependent on the events proceeding in the nucleus. We do not, for instance, find a cell cleaving before the nucleus has entered into its division process, or a cell membrane cutting through a spindle before the separation of the chromosomes has occurred. The dependence of cell division on nuclear phenomena is, however, not absolute, since it can occur in parts of cells which contain no nucleus (p. 64). But in these anucleate cells, centrosomes, if missing, arise de novo, and it seems that they control the occurrence of the cleavage; thus even in this case the cytoplasmic division is not wholly independent of the behaviour of the achromatic apparatus.

The nature of the connection between the nuclear division and the cleavage of the cell body is not well understood. Swann (1951, 1952) has showed by studies with the polarising microscope that the orderly arrangement of the material which forms the asters at each pole of the spindle decreases as the chromosomes come into their neighbourhood at anaphase. He suggests that this is brought about by a substance released from the chromosomes, and that this substance diffuses away from the two daughter-nuclei until it reaches the cell cortex, where it initiates the processes leading to cell cleavage. It would seem that some diffusing agent of this kind must almost certainly be involved, but it is not clear that it arises from the chromosomes.

Darlington (1937) has drawn attention to the type of cell cleavage which occurs in cases where the movement of some of the chromosomes on the spindle has been abnormal. He claims that if one or two chromosomes have lagged behind the others and become included in a small separate nucleus of their own, a plane of cell division often forms around this nucleus, but only in those cases in which the chromosomes are provided with centromeres: the cleavage planes pay no attention to acentric fragments. Again, it is well known that if two homologous chromosomes become joined together (e.g. by chiasmata inside an inversion) and are unable to separate properly at anaphase, they form a ‘chromosome bridge’ connecting the two telophase nuclei, which move away from one another as far as the connection will allow. The cleavage planethen forms between the two nuclei but is unaffected by the chromosome bridge and cuts through it as though the connecting chromosome material were not present. From this type of evidence Darlington concludes that it is the centrosomes and centromeres which affect cell cleavage rather than the chromosomes themselves.

The first factor, then, which plays a part in the determination of the cleavage pattern of an egg is the orientation and position of the cleavage spindles which initiate the changes in the cell body. In spirally cleaving eges, for instance, from the 4-cell stage onwards the cleavage spindles are obliquely inclined, first to one side and then to the other of the vertical, and this gives rise to the characteristic pattern of the group of cells. The nature of the factors which in their turn determine the orientation of the spindles is unknown, but there must be some sort of continuous change proceeding within the cytoplasm which controls their development. The occurrence of such changes is well shown by some experiments of Horstadius (1939) on the echinoderm egg. He used eggs of the seaurchin Paracentrotus lividus, which possesses a sub-equatorial band of pigment, which makes it possible to recognise the orientation of the egg even when the cleavage is abnormal. By treatment with hypotonic sea water or by shaking, one can cause a delay in the appearance of the cleavage furrows, but the results show that the factors controlling the orientation of the spindles go through their usual changes at the normal rate. Thus the first cleavage may be delayed until the spindle mechanism is ready for the second cleavage, and the next until it is intermediate between the normal second and third (Fig. 4.4, second row).

This same experiment also shows that the orientation of the spindle is not the only factor concerned in the cleavage pattern. In the Paracentrotus egg the vegetative region after a certain time has a tendency to form very small cells (micromeres), and will do so whatever the orientation of the spindle which initiates the division (Fig. 4.4, row 3). Again, spontaneous constrictions, mimicking the early stages of cleavage, are seen in the parthenogenetic eggs of Chaetopterus which ‘differentiate without cleavage’ (p. 63), and in isolated and non-nucleated polar lobes of molluscs (Morgan 1933). The operative agent in these instances is almost certainly located in the egg cortex.

File:Waddington1956 fig4.4.jpg

Figure 4.4.

Delay of cleavage relative to the orientation of the spindles. Upper row, the normal cleavage of the echinoderm Paracentrotus. Middle row, cleavage somewhat delayed. Lower row, cleavage further delayed, the first cleavage not occurring till the spindle is orientated vertically. (From Hérstadius 1937.)

There are many other instances in which it can be shown that the local properties of the cortex influence the course of the cleavage planes. This factor is of importance in nearly all eggs in the formation of the small polar bodies. Morgan (1937) has tried to discover why the maturation spindles in the egg normally give rise to such extremely unequal divisions of the cell body. He showed that in the marine snail Ilyanassa the second polar-body spindle could be shifted into the middle of the egg by centrifuging, and that in this position, when it is no longer near the polar cortex, it is capable of causing the egg to divide into more or less equal parts. It only does so if the egg is still somewhat elongated after the centrifugation; if the egg becomes completely rounded up again, a centrally-placed maturation spindle fails to cause it to divide. In the parthenogenetically activated Urechis eggs studied by Tyler (p. 54), the displaced polar-body spindles seem to be more effective and able to cause an equal division even of a spherical egg, provided they have been shifted away from the polar cortex.

Another clear example of the influence of the cortex is provided by the experiments of Lehmann (1946) on the freshwater oligochacte Tubifex. In this form, considerable protuberances are pushed out from the body of the cell, both at the first and second polar-body divisions and at the first cleavage division (Fig. 4.5). In the polar-body divisions, which occur with the spindle near the animal pole, the protuberances are arranged in a more or less symmetrical manner around the animal-vegetative axis. The first cleavage division is unequal and gives rise to a small AB cell and a large CD cell. The protuberances at this division form mainly at the equator of the cell, in particular in the region of the AB blastomere. They are thus arranged bilaterally symmetrically as seen from the animal pole. By moderate centrifugation the spindle of the second polar-body division can be moved from its normal position without the structure of the cortex being materially affected. If the egg is arranged so that the centrifugal force is parallel to the animal-vegetative axis, the internal contents of the cell are stratified and the polar-body spindle moved from the animal pole into the interior. It is found that the pattern of the protuberances which form at the next cell division is hardly altered, even if the spindle has been moved right down to the vegetative pole of the egg. If, however, the egg is orientated so that the centrifugal force is at right-angles to the axis, the spindle is shifted towards the egg equator on one side. With this orientation the egg becomes much more elongated, with a given degree of centrifugation, than it does when the force acts along the egg axis; this presumably indicates that the egg cortex is more easily deformed in the equatorial than in the animal-vegetative plane. When second polar-body formation begins, protuberances appear in the equatorial region of the egg in the neighbourhood of the spindle. They thus form a bilaterally symmetrical pattern very similar to that characteristic of the normal first cleavage, in which again it is an equatorial region of cortex which is closest to the initiating spindle. It is clear, then, that the protuberances are produced by an interaction between the cleavage spindle and the cortex in its neighbourhood, which has a structure which differs in the different parts of the egg. Lehmann claims that this structure is to some extent visible in the living egg, which shows a pattern of nine to fifteen subcortical meridianal thickened strands of the heavy fibrillar type of cytoplasm which he names ‘plastin’. This material is, however, at least to some extent, shifted by the centrifugation, and the structure which persists in the cortex of the centrifugal eggs is perhaps not due to the plastin, but to some associated structure in the cortex itself.

File:Waddington1956 fig4.5.jpg

Figure 4.5.

Maturation and first division of the egg of Tubifex, seen from the animal pole; a, soon after laying; b, formation of protuberances during the extru sion of the first polar body; ¢, between first and second polar body divisions; d, extrusion of the second polar body; e, before first cleavage of fertilised egg (the animal pole plasm shaded); f, early stage of cleavage, pole plasm elongated; g, h, stages of cleavage showing protuberances; i, two cell stage (the AB blastomere above, CD below). (After Woker 1944.)

There is normally also an accumulation of plastin around the nucleus. With mild centrifugation in a polar direction at a stage shortly after fertilisation, all the plastin is driven to the centrifugal end while the nucleus still remains near the centripetal end, surrounded only by yolky cytoplasm. In such eggs the nucleus shows no sign of entering into division, which presumably indicates that the nuclear processes are normally initiated by a reaction which involves the surrounding plastin material. Further, no deformations of the cell cortex occur, which again demonstrates that they are initiated by the nuclear division process. These interactions between cortex and nucleus in Tubifex are somewhat reminiscent of those between the nucleus and the cortical granules shortly after fertilisation in the echinoderms, as described by Allen (p. 51).

It appears, then, that the pattern of cleavage is determined by interactions between the spindle and the cortex. We have now to consider the nature of the forces which bring about the deformation of the cell and its division into two. There are a number of theories in the field. As the first group we may take those which suppose that the nuclei and spindle apparatus continue to play in important part throughout the whole course of the division. For instance, Gray (Review: 1931), from studies on the sea-urchin egg, suggested that the asters continue to grow until all the available cytoplasm is incorporated into two spheres of radially arranged material at the poles of the spindle, and that the cortex passively accommodates itself to these two masses, which form the two first blastomeres. Dan (Review: 1948), working on the same form, suggests that the astral rays are actually attached to the cortex, and he shows by means of a model how, if this were so, a contraction of the rays would cause the bending in of the cortex and at least the beginning of the process of division. However, these mechanisms certainly do not operate in all types of eggs, if indeed in any. For instance, cleavage in the amphibian egg can continue quite satisfactorily when a large amount of the internal contents has been removed so that the cortex is quite flaccid and the asters unable to produce any internal turgor. Moreover, after a cleavage furrow has begun to form it can extend over a region of cortex which has been isolated from the spindle by the insertion of a strip of cellophane, which must certainly prevent the attachment of any astral rays (Waddington 1952d; Mitchison 1953 has similar evidence in echinoderms). (Fig. 4.6.) Finally Swann and Mitchison (1953) find that if sea-urchin eggs are treated with colchicine at anaphase, when the chromosomes have separated but the division of the cell body has not yet begun, the asters disappear but the cleavage occurs normally. Thus it seems fairly certain that although the division spindle initiates the process of cleavage it does not play any straightforward mechanical part in carrying the process through to completion. It seems that the main active agent must be the cortex itself.

Probably the most widely accepted theory is one which ascribes the cleaving of the cell to the contraction of a ring of cortex around the position of the future furrow. This theory, which has recently been defended by W. H. Lewis (1951) and Marsland (1951, Marsland and Landau 1954) is the one which suggests itself most naturally when one looks at dividing cells and tries to think how their behaviour might be explained in terms of cortical activity. There is, however, rather little direct evidence for it. And, as Mitchison (1952) points out, there are some difficulties in it when one examines the matter more closely. In the first place, if the cell is to be constricted completely into two parts, the ring of contracting material would have to contract away to nothing. This, Mitchison argues, would seem to be an unlikely event, if one thinks of it in terms of a contraction like that of muscle: it is perhaps not so unplausible if one pictures the contracting ring as similar to the ectoplasm at the posterior end of an advancing amoeba, which liquefies after it has finished contracting. Again, when a cell divides its surface area must increase; in the case of a spherical egg becoming converted into two spheres, the increase is about 26 per cent. A contraction of the ring of the furrow would therefore have to be compensated by a large increase in area elsewhere. The main evidence on which Marsland relies, for support of the ‘contracting ring’ hypothesis, is the demonstration that if gelation of the cortex is prevented (by high hydrostatic pressure or low temperature) the development of the cleavage furrow is inhibited. This might be due, as he suggests, to an abolition of the contraction; but it might equally be a consequence of the failure of the ungelated cortex to transmit the stress from the expanding regions to the furrow in the way required in the mechanism postulated by Mitchison and Swann. Thus the evidence is not fully conclusive. Mitchison and Swann (1955, see Mitchison 1952) have recently turned the conventional theory upside down and suggested that the prime mover in cell cleavage is not a contraction of the furrow region but an expansion of the other parts of the cortex. They suppose that the cleavage process is initiated by substances released by the two separating groups of chromosomes (there seems no reason why one should not attribute the activity to the centromeres, rather than to the chromosomes themselves, in accordance with the points made on p. 66). This substance would diffuse out into a more or less dumbbell-shaped region which, in a spherical egg, would reach the cortex first at the equator opposite the poles of the spindle (Fig. 4.7). The substance is supposed to cause an expansion of the cortex, and this would begin in the same region. Meanwhile the ring of cortex which lies in the plane of the equator of the spindle (and therefore in Animal pole includes the poles of the egg) will become flattened for two different reasons. Firstly, Mitchison claims that there seems always to be some tension in the egg cortex, so that when it expands in one place, the other regions will tend to contract; and secondly, the expansion of the cortex on the egg equator is supposed actually to push the rest of it towards the plane of the spindle equator'. These two processes will, in fact, cause the future furrow not merely to flatten but to bend inwards. This will bring it in contact with the expanding dumbbell-shaped area containing the diffusing active substance, and when this happens the cortex in the furrow will be acted on by the substance and caused to expand; and this expansion completes the formation of the cleavage plane.

File:Waddington1956 fig4.6.jpg

Figure 4.6.

The independence of the cleavage furrow, once it has started, of contact with the spindle. In a newt’s egg which was just starting to cleave at 3.23 p-m., a strip of cellophane was inserted under a region through which the furrow should extend. Within the next half-hour it did actually extend through the area. (From Waddington 19524.)

File:Waddington1956 fig4.7.jpg

Figure 4.7. Diagram of cleavage in the sea-urchin. (After Mitchison 1952.)

This theory was worked out in the first place from studies on echinoderm eggs. In that form there is a fair amount of support for various parts of it. Thus Mitchison and Swann have presented evidence that the cortical changes leading to cleavage begin in the equatorial regions of the egg, opposite the poles of the spindle, in the form of a decrease of the birefringence and light-scattering properties of the cortical material. Although the structure of the cortex is not at all well understood, it is reasonable to suppose, as Mitchison suggests, that it is made up largely of protein chains folded at right-angles to the surface, and that the changes in birefringence are connected with an expansion in area. Again, Dan and Ono (1954, see Dan 1948, 1954) have followed the movements of small particles of kaolin attached to the cell surface. They found that there was an expansion starting opposite the poles of the spindle and spreading towards the future furrow. The furrow region itself at first contracts somewhat, and then expands greatly as the new plane of division cuts down into the depth of the cell.

Finally, Mitchison and Swann (1955) have devised an apparatus for measuring the deformability of the cell surface. The tip of a small pipette is brought against it and a negative pressure or suction applied; one can then measure the height of the small protuberance which the suction raises on the surface. With this apparatus they showed that shortly before division the cortex becomes much less easy to deform. They suggest that this is not due to an increase in a tension (such as a surface tension) in the cortex, but rather to that layer becoming stiffer or less plastic. They argue that this makes it easier to suppose that the expanding regions opposite the poles of the spindle can successfully push the rest of the cortex into the furrow region.

1 The student is warned to beware of the verbal pitfalls that can occur from the fact that the long axis of the first cleavage spindle is perpendicular to the animal-vegetative axis of the egg.

Raven (1948) has estimated the ‘tension at the surface’ of the eggs of Limnea from fertilisation till first cleavage. His method was to centrifuge the egg for 5 minutes at a moderate speed, which gave a force of 1860g. Observation of the distinctness with which the internal contents become stratified gives an indication of the viscosity of the cytoplasm, while the extent of the elongation of the egg allows one to draw some tentative conclusions about the deformability of the surface. The elongation will, however, also be affected by the degree to which the internal constituents separate into definite layers, so that the viscosity changes somewhat obscure the picture of the alterations in the properties of the surface. Raven finds that the viscosity is low immediately before each of the first three cleavages, but rises as the furrow makes its appearance, and reaches a maximum about 10-15 minutes later. The data on the surface tension are not so clear cut, and only the first cleavage has been investigated. Raven finds that it is low immediately before the appearance of the furrow. His diagrams show, although he does not refer to the fact in discussing them, that it rises steeply during the cleavage. It is not quite clear whether this rise occurs at a slightly later period than the similar increase in stiffness of the cortex of the echinoderm egg, but, apart from possible minor changes in timing, the phenomena seem rather alike in the two groups.

These results are good evidence that some, at least, of the processes envisaged in Swann and Mitchison’s theory actually occur. Moreover there is plenty of other evidence that expansions of the cell membrane are often associated with cleavage. Some of the most extreme examples of this are scen in those spirally cleaving eggs in which a polar lobe is formed (Fig. 6.2). But the mere occurrence of an expansion does not suffice to show that it is the prime mover in bringing about a cleavage of the cell, and there is some reason to doubt whether it is more than one out of a number of factors which may play a part. For instance, we have seen that in Tubifex there are considerable cortical expansions which cause the egg to throw out protuberances at the times of polar-body division and at the first cleavage division. The second cleavage division occurs first in CD blastomere and only somewhat later in the AB one, and is not accompanied by such a pronounced cortical expansion as the earlier ones, though the CD cell does elongate considerably and increase its surface area to a fair extent just before the cleavage occurs. Huber (1947) has studied the action of two anti-mitotic substances, naphthoquinone and phenanthrenequinone. He finds that the former has a particularly strong effect on the expansions, tending to suppress the formation of protuberances in the first division and the stretching of the CD cell in the second. The second substance, on the other hand, leaves these processes relatively unaffected or even exaggerates them, but the cells tend to fail actually to divide into two, so that one finds, for instance, an elongated but undivided CD cell (Fig. 4.8). Huber therefore concludes that cell division involves two different cortical movements, not only the expansion seen in the formation of protuberances, but also a contraction in the furrow region.

A consideration of more yolky types of egg would also suggest that a mere expansion of the already existing cell cortex cannot be the only factor causing the cleavage. In the extremely yolky eggs of a sturgeon, for instance, the cleavage furrows eventually extend a long way from the position of the spindle, and it is difficult to see how the influence of a cortical expansion starting in the neighbourhood of the spindle poles could reach so far (cf. Fig. 4.1). Moreover in such forms the second cleavage furrow starts to form in the animal region while the first furrow is still working its way down towards the vegetative part of the egg. It seems unlikely that any system of expansions working through the cortex as a whole could control the furrows in such cases. It would be much easier to attribute the formation of the cleavage planes to factors located in the immediate neighbourhood of the furrows themselves.

File:Waddington1956 fig4.8.jpg

Figure 4.8. The effect of quinones on cleavage: a, section through a 2-cell stage of the egg of Tubifex; the nuclei are already in telophase of the second division and the CD cell (below) is markedly elongated preparatory to dividing in two; b, a similar stage from an egg treated with 1,4~-Naphthoquinone, note the slight elongation of the CD cell; c, after treatment with 9,10-Phenanthrenequinone the elongation of the CD cell is exaggerated. (After Huber 1947.)

Studies on the moderately yolky eggs of the Amphibia suggest, indeed, that in them also cortical expansion is not such an important factor in cleavage as Mitchison and Swann suppose it to be in the smaller eggs of the echinoderms. Selman and Waddington (1955) have shown that there is little movement of cortical pigment granules in the neighbourhood of the poles of the spindle and there is no noticeable cortical expansion there. However there is a considerable flow of the granules along the furrow as it first appears near the animal pole of the egg. This movement along the furrow would seem to indicate a contraction acting in this direction and at this place. Moreover if the eggs are viewed from the side it can be seen that just before division the whole cell heaps itself up so that its vertical height increases (Fig. 4.9). This movement is accompanied by an increase in the resistance of the cortex to deformation as measured by the suction apparatus of Swann and Mitchison. This must involve a force acting in the cortex, which might be either analogous to a surface tension, or, more probably, due to an increase in the elastic constants of the material. In any case, the rising up of the egg suggests a decrease in surface area rather than an increase. It becomes rather unplausible, then, to attribute much importance to cortical expansion in this case; we seem rather to have to deal with a localised contraction along the length of the developing furrow.

File:Waddington1956 fig4.9.jpg

Figure 4.9. Side views of cleavage in a newt’s egg removed from its vitelline membrane to show the ‘rounding up’ at the beginning of division. A, part-way through the first division; B, between first and second division; C, beginning of second division; D, second division completed. (After Selman and Waddington 1955.)

There is, however, almost certainly another factor involved. When an amphibian egg cleaves, the surface between the two blastomeres is almost unpigmented, and thus differs sharply from the cortex of the uncleaved egg. It seems that it must have arisen de novo. Schechtman (1937) suggested that it is formed by the growth of the cortex into the depth of the furrow. Mitchison and Swann argue that such growth takes place, not merely from the very edge of the original cortex, but by intussusception of new material throughout the whole area of the infolding region; if this were so, the process becomes in effect a type of cortical expansion.

A similar increase in surface area occurs in the furrow region of the echinoderm, but in this form it seems to take place after the cleavage plane has cut through the egg (Fig. 4.10). According to Dan (1954) it also seems to be due to intussusceptive growth, accompanied by stretching. From this evidence, Mitchison and Swann conclude that the cortical expansion involved in their theory always includes an element of intussusceptive growth. But even if this is so, the amount of growth as compared with mere stretching must be so vastly greater in the furrow region than elsewhere as to amount to a qualitatively different type of phenomenon. Moreover, Selman and Waddington find that sections of an amphibian egg in the process of division show that an indication of the future cleavage plane is developed in the cytoplasm over a much wider area than corresponds to the externally visible furrow. It seems very unlikely that the material making up this precursor of the division plane is directly derived from the original cortex. It looks rather as if it differentiates in situ (although it must be the cortex which initiates the process of differentiation). It gradually increases in extent, and also in thickness, and eventually splits into two separate films which form the outer membranes of the two blastomeres in the region where they are in contact. If the vitelline membrane is removed, so that there is nothing to hold the two cells together, they tend to fall apart as they settle down on the bottom of the dish, and the white newly formed cell membrane is then extensively exposed in the depths of the furrow as one looks at the egg from the animal pole; it may be visible within the furrow even in eggs cleaving inside the membrane.

File:Waddington1956 fig4.10.jpg

Figure 4.10. A. Section through part of a newt’s egg at an early stage in the first cleavage. The future surface which will separate the two blastomeres is already indicated in the internal cytoplasm although there is not yet any furrowing of the external surface in this region. (After Selman and Waddington 1955.) B. Three stages in the cleavage of an echinoderm egg. Small kaolin particles (indicated by short lines) have been placed on the surface. Note that they at first move into the furrow (2), but that later new cortex is produced in the depth of the furrow (dotted), so that the particles move out again. (From Dan 1948.)

The evidence available at the present time would seem, therefore, to suggest that several factors are operative in the process of cell cleavage, their relative importance differing in various groups of animals. These factors are: firstly, localised expansions of the cortex; secondly, an increase in stiffness of the cortex; thirdly, in the Amphibia at least, an increase in the tangential force acting in the cortex; fourthly, a contraction localised in the neighbourhood of the furrow and directed along its length; fifthly, a formation of new cell membrane from the sub-cortical cytoplasm. It is possible that Swann and Mitchison are correct in ascribing the major importance to the first two of these in the echinoderm egg but it scems likely that in other eggs, particularly those with large quantities of yolk, the other three factors have to be taken into account.

Suggested Reading

Fankhauser 1948, Mitchison 1952, Swann 1952, Lehmann 1946.

   Principles of Embryology (1956): Part 1 - 1 The Science of Embryology | 2 The Gametes | 3 Fertilisation | 4 Cleavage | 5 The Echinoderms | 6 Spirally Cleaving Eggs | 7 The Ascidians and Amphioxus | 8 The Insects | 9 The Vertebrates: The Amphibia and Birds | 10 The Epigenetics of the Embryonic Axis | 11 Embryo Formation in Other Groups of Vertebrates | 12 Organ Development in Vertebrates | 13 Growth | 14 Regeneration | 15 The Role of Genes in the Epigenetic System | 16 The Activation of Genes by the Cytoplasm | 17 The Synthesis of New Substances | 18 Plasmagenes | 19 The Differentiating System | 20 Individuation - The Formation of Pattern and Shape | References
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