Talk:Waddington1956 6

From Embryology


THE SPIRAL type of cleavage is sufficiently definite to allow one to recognise the existence of a common pattern in several groups of animals which are not otherwise very closely related (molluscs except cephalopods, nemerteans, platyhelminths, annelids); but overlying the basic similaritics there are very many variations in detail, both in the early cleavages and in the types of larvae which are produced by early development. To keep within the limits of space available here, it will be necessary to attempt a ruthless simplification.

The cleavage begins by two more or less vertical divisions, cutting the egg into four blastomeres, which are typically somewhat twisted in relation to each other. These four are conventionally known as A, B, C, and D. In the next few cleavages, each of these cells remains as a fairly large macromere, and gives off a succession of smaller micromeres, the first group of which are known as 1a, 1b, 1c, 1d, the second group as 2a, 2b, 2c, 2d and so on. The cleavage spindles by which these divisions occur do not lie either vertically or horizontally, but at some angle between; and if the spindles are tilted to the left of the vertical in the formation of the first group, they will be tilted to the right for the next group. There is in fact an alternation from one tilt to the other. While the macromeres are giving off new rings of micromeres in this way, the already formed micromeres continue to divide in the normal way, again with their spindles tilted like those of the macromeres (Fig. 4.2, p. 61).

This regular pattern usually continues for four division-cycles. The subsequent fate of each of these cells has been followed in detail, and we know exactly what organs each will form in the later embryo. There is no need to go into great detail about this here. Roughly, the macromeres form endoderm, and most of the micromeres ectoderm; but there are two special micromeres which produce the mesoderm, and which we shall find play a peculiarly important role in the mechanics of development. These are the cells 2d and 4d, both of which, as their designation indicates, are ultimately derived from the D macromere.

Events after the initial cleavage vary a great deal in different groups. In many species a larva is formed; and this is usually some variety of the ‘trochophore’ type (Fig. 6.1). In the development of this, the ectodermal micromeres grow down over the macromeres, which bend inwards to form


a pocket-shaped primitive gut, which gradually elongates until the whole embryo assumes a shape like an old-fashioned peg top. There is usually an apical tuft of cilia, and a strong ciliated band (the prototroch) more or less at the equator. Meanwhile the 4d mesoderm cell has been covered over by ectoderm, lying in the corner at the boundary between ectoderm and endoderm. It divides into two, and each of these daughter-cells gives off a series of endomesoderm cells. In molluscs, these again become scattered, but in annelids they remain joined together as two long bands.



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Gastrulation and formation of the trochophore larva in spirally cleaving eggs. A showsa section through a late cleavage stage, the micromeres lying directly on top of the larger macromeres, the slight space between them corresponding to the cavity of the blastula. Some cells from 2d and 4d (shaded) have sunk beneath the surface. B, the micromeres spread over the macromeres, which are being drawn up into the embryo, forming the beginning of the primitive gut. C is an early trochophore; note the two large cells, derived from 4d, each of which is budding off a row of mesoderm cells which extends round the blastopore between the ectoderm and endoderm. D, the trochophore is beginning to elongate, and the mesoderm bands are swung into a vertical position (this is typical of certain worm embryos, such as those of Polychaetes). A mouth has appeared, either as a new opening where the primitive gut has broken through the ectoderm, or in some forms by the blastopore becoming constricted into two. 96 PRINCIPLES OF EMBRYOLOGY

These at first extend round the gut which is being pushed in from the blastopore, but as the whole embryo elongates, the mesoderm bands come to lie more or less vertically. Finally a mouth is formed, either by the tip end of the gut breaking through the ectoderm to the exterior, or in some forms by a constriction which divides the blastopore into two. A trochophore larva of this kind is obviously only a minor variation from the general structure of a gastrula, and the whole egg takes part in its formation.


Direct development (without larval stage) in a spirally cleaving egg (the freshwater oligochaete Tubifex). The macromeres have cleaved to give a compact mass of largish endoderm cells (Ex), while most of the micromeres have formed small ectoderm cells lying on the other side of the egg (Ek). The cell 2d has, however, produced two groups of ‘stem-cells’, each comprising one neuroblast and three secondary myoblasts; these bud off a row of neural cells and three rows of ectodermal muscle cells. Below each of these columns of four are mesoderm-stem cells derived from 4d, which produce the accompanying mesoderm. Each column is known as a halfgerm-band (rk and /k); they unite on the other side of the embryo to form the head, and, as they grow, to give rise to the rest of the body. (After Penners.)

In these forms with larval stages in their development, the formation of the adult does not take place till considerably later; we shall not discuss the processes by which this eventually occurs except to point out that the ectoderm and mesoderm of the adult ultimately trace back to 2d and 4d. In some spirally cleaving types, such as oligochaetes, the development of the egg is ‘direct’, in that the adult is formed without passing through any special larval stage. In these animals it is difficult to find anything which can be called a normal gastrula stage. The ‘somatoblasts’ 2d and 4d each divide into a right and a left half; and the cells so formed become the mother-cells from which a long string of daughter-cells is budded off. SPIRALLY CLEAVING EGGS 97

The cells derived from 2d form ectoderm, and overlie those from 4d which form mesoderm. Thus two bands are produced, one on the right and one on the left, both lying on the surface of a mass of cells derived from the A, B and C quadrants, and each consisting of a core of mesoderm covered by ectoderm. The mother-cells continue to divide, and as these bands elongate, they push out over the surface until they meet and fuse; from the fused bands the adult worm develops. Thus in these eggs, the whole embryonic portion is formed from 2d and 4d, the rest of the cells taking little part.

A very large number of experiments have been made on spirally cleaving eggs, but rather little insight has been gained into the factors which control their development. In general, it is found that from a very early stage isolated blastomeres behave as though they were still part of a complete egg, and develop only into those organs which they would have formed if left undisturbed (Review: Schleip 1929). This fact gave rise to the suggestion that there is, in these eggs, a strict localisation of “organ-forming areas’, or regions each of which could develop only into certain definite organs. The egg was considered to be a mosaic of such areas, and such ‘mosaic’ development was contrasted with the ‘regulation’ development found for instance in echinoderms.

We now know that the mosaic character is by no means absolute. As an example which demonstrates both the truth and the falsity of the idea, we may consider the egg of the nemertean Cerebratulus. Hérstadius (1937) has made some isolations and recombinations of the various rings of cells at the 16-cell stage, quite comparable to his experiments on echinoderms described on p. 85. In Cerebratulus he found completely ‘mosaic’ behaviour; each ring of cells, when isolated, formed only what would be expected of it, and in combinations there was no sign of interaction between the animal and vegetative groups. But if one goes back to a slightly earlier stage, things are not quite the same. Any nucleated fragment cut off from the unfertilised egg, can, after fertilisation, develop into a normal embryo. And even though the differences along the animal-vegetative axis are fixed as early as the 8-cell stage, those in the other plane are certainly labile as late as the four cell, since the isolated first four blastomeres may each give a normal larva. Thus there must be some process of determination which gradually fixes the manner in which the parts of the egg can develop; but, since this is complete by the 8-cell stage, it must go much faster than in echinoderms, where we saw that regulation is possible considerably later; and unfortunately in the mosaic eggs we know of nothing which controls development in a way comparable with the animal and vegetative gradients (Fig. 6.3). 98 PRINCIPLES OF EMBRYOLOGY

In many of the spirally cleaving eggs, the determination of future development is complete (or nearly complete) even earlier than in Cerebratulus. We have seen that the 2d and 4d cells are particularly important especially in species with no larval stage; and it is often found that the D quadrant is already different from the others from the very


Mosaic development in the nemertean worm Cerebratulus. A and B show the normal 8- and 16-cell stages. C is the normal ‘pilidium’ larva (a variety of the usual trochophore larva). D is a relatively normal pilidium from an isolated blastomere from the 2-cell stage. E, the same from an isolated blastomere of the 4-cell stage. F, larva from isolated four animal cells from 8cell stage; note the absence of any gut and the exaggeration of the apical tuft. G, larva from four vegetative cells isolated at 8-cell stage. H, middle two layers (an, and veg;) isolated from 16-cell stage. I, larva from combined an, and veg, from 16-cell stage. Note the failure of regulation in H or of interaction in I. (From Hérstadius 1937.)

beginning of development. Thus in the oligochaete Tubifex (Review: Lehmann 19482) only the cell containing the D quadrant will develop any embryonic structures if the first two or the first four blastomeres are isolated. There is therefore already something in the D quadrant which is necessary for the formation of an embryo. But this does not mean that all the details of development have been completely fixed by this time. SPIRALLY CLEAVING EGGS 99

Perfectly normal embryos are formed if A, B or C quadrants are eliminated, and this necessitates some replacement of them by converting part of the D substance for the purpose. Similarly the macromere 4D can be dispensed with. Even if the individual somatoblasts are killed at the stage when the germ-bands are beginning to form, some regulation is still possible. For instance, if the mother-cells of the two ectoderm bands are killed, the embryo has at first no ectoderm, but some is later formed by a conversion of mesoderm cells. If the mesoderm mother cells are killed the mesoderm is not produced from any other element; but it is found that there are characteristic defects in the development of the uninjured ectoderm, which points to the existence of essential influences of mesoderm on ectoderm in normal development (Penners 1938). Thus the mesoderm seems to have in some sense a leading role in the whole embryogenesis; we shall find a much more striking example of the same thing in vertebrates.

The essential substance of the D quadrant can sometimes be seen, in the form of a special type of cytoplasm. In the mollusc Dentalium, for instance, the cleavage is very oddly modified in connection with a region of clear cytoplasm lying near the vegetative pole. Before the first division, this material is pushed out from the egg in a broad pseudopodium-like lobe. The cleavage plane runs in such a way that the whole of this gets into one of the two daughter-cells; when the lobe has been retracted and the whole cell rounded up, this blastomere is considerably larger than the other. A similar process occurs in the succeeding division, and most of the material of the lobe eventually gets into the D blastomere (some may be included in C). One of the early and classical experiments on spiral eggs is that of E. B. Wilson, who showed that when the polar lobe is removed the development of the whole embryonic region (derived from 2d and 4d) is suppressed (Fig. 6.4). Perhaps more surprising is a recent result (Novikov 1940, on Sabellaria); by treatment with KCl, the first cleavage can be made to become equal, so that the polar lobe substance get into both the first two blastomeres instead of only into one. Twin embryos are formed. This must involve a considerable amount of regulation, so here the lobe material has acted, not merely as a region of the egg whose fate has been determined precociously, but as one which can initiate embryonic development by the cells surrounding it. Other agents (c.g. abnormal temperatures, anaerobiosis, etc.), may upset the position of the first cleavage spindle in many types of spirally cleaving eggs, with the result that twins are produced (Tyler 1930).

We therefore seem to be confronted with the situation that, in the so-called mosaic eggs, there is at a very early stage (which varies from 100 PRINCIPLES OF EMBRYOLOGY

before fertilisation till the time of the second or third cleavage) a localisation of types of cytoplasm which control the direction of later development; but one of these substances, namely that which normally gets into the D quadrant, can in some species cause a considerable amount of reorganisation to go on in its neighbourhood, so that in eggs of this type a whole embryo tends to be formed around it.


The polar lobe in the mollusc Dentaliuim. In the uncleaved egg (A) there are specialised odplasms at both animal and vegetable pole. At the first division (B), the vegetative pole plasm is protruded as a ‘first polar lobe’ (S,). After the division this is withdrawn into the CD blastomere, whence it protrudes again as a ‘second polar lobe’ (S.) at the second division D, after which it is withdrawn once more into the D blastomere. At E is shown the normal trochophore larva; at F and G the defective larvae produced when the first (F) or the second (G) polar lobe is removed. (After Wilson.)

Recent investigations have tended to concentrate on two problems; to discover more about the nature of the substances whose localisations are responsible for the mosaic aspects of development in these forms, and to try to find out the factors which bring about the localisation. Some progress has been made in both directions.

Local differences in the cytoplasm, which are invisible in Portal life, can sometimes be revealed by treatment with suitable dyes or histochemical reagents. Vital dyes which act as pH or rH indicators (such as SPIRALLY CLEAVING EGGS IoI

Janus Green, Neutral Red, etc.) often show different colours in the different regions of mosaic eggs; usually these differences are related to the main animal-vegetative axis. In typical regulation eggs, such as echinoderms, the differences are absent or at least much less well developed, but the same is also true in some mosaic eggs, where they would be expected. It is not easy’to give an exact interpretation of the phenomena, since it is notorious that when indicator dyes are in the presence of protein, their behaviour is atypical, and one cannot simply deduce the pH or tH from the colours which they take up. Nevertheless, the existence of differences in colour certainly demonstrates the presence of some differences or other in the cytoplasm, even if it is unsafe to conclude much as to their nature. Histochemical tests for various enzymes (e.g. indophenoloxidase), or fixed -SH groups, ascorbic acid, etc., have also revealed certain cases in which these substances are strictly localised within mosaic eggs. The subject has recently been reviewed by Needham (1942, p. 131 seq.) and Brachet (1944, p. 271, seq.). It will be seen from their discussions that the biochemical interpretation of the findings is not clear in this instance also. In particular, the suggestion of Ries (1942), who has been one of the most active workers in this field, that the most important differences between the regions of mosaic eggs are related to the intensities of respiration, is almost certainly based on too optimistic a neglect of the possible sources of uncertainty. Nevertheless it is important that a beginning at least has been made with the biochemical recognition of cytoplasmic localisations.

It has also been possible to obtain some information about the physical properties of the substances involved in the mosaic regions. One may ask whether they are small-molecular substances, such as amino-acids or vitamins; or on the other hand are they larger entities which should be regarded as cell constituents rather than chemical molecules? The evidence which has been available for some time, that they can be shifted about within the egg-cell by centrifugation, provides grounds for preferring the second alternative. Recently Lehmann and Wahli (19 54) have made a careful study with the electron microscope of the structure of the cytoplasm in the various blastomeres of Tubifex. They find that at a fairly early stage there are characteristic quantitative differences between the cells. Thus by the time the four sets of micromeres have appeared, the cell 2d (which will produce the embryonic ectoderm) has more of the basophilic fibrillar cytoplasm, with many small globular particles and peculiar spindle-shaped bodies, whereas 4d (which gives the embryonic mesoderm) has sparse fibrillar cytoplasm, few of the globular or spindle-shaped bodies but many of a larger type of particle which can be designated 102 PRINCIPLES OF EMBRYOLOGY

as ‘mitochondria’. These differences, which it must be remembered affect only the concentration of the particles, none of which are completely absent in any region, cannot be seen in the newly fertilised egg, which contains a population of microsomes which appears uniform in the electron microscope. It seems most probable, however, that these microsomes differ in their chemical properties, and that it is the microsome population which determines the character of the ooplasms.

The second problem which has been attracting attention recently is that of the mechanisms of localisation. The original distribution of materials in the egg can be fairly easily disturbed by centrifugation, as a consequence of which the egg contents become stratified into layers of different specific gravity. As would be expected if the eggs behaved in a strictly mosaic manner, this stratification frequently leads to the production of abnormal embryos, But this is not always the case; almost perfectly normal larvae may develop from eggs which have suffered a severe stratification. It used to be thought that the explanation of this must be that the substances which become stratified are not those which are morphogenetically active, but comprise only relatively neutral materials such as yolk. However, the application of histochemical tests has demonstrated the presence of several important enzymes in the stratified layers, and it therefore becomes rather unplausible to advance this hypothesis. Raven (1948) has made a particularly careful study of the phenomena in the snail Linnea. He showed that in the unfertilised egg there is a visibly differentiated ‘sub-cortical plasm’ located near the vegetative pole, and that soon after fertilisation an ‘animal plasm’ appears near the animal pole. These two plasms move in definite ways during the early stages of development; before the first cleavage, the sub-cortical plasm spreads upwards so as to clothe the entire surface of the egg just below the cortex, while the animal plasm also extends somewhat, but eventually comes to lie mainly in the micromeres (Fig. 3.3, p. 49). If an egg is centrifuged in such an orientation that the pole plasms are moved away from their normal location, it is found that they very soon make their appearance again in their original positions. The rapidity of this distribution differs according to the exact stage when the centrifuging is carried out, since the viscosity of the cytoplasm varies throughout the progress of the cleavage. Raven claims that the redistribution of most substances can continue, even after the appearance of the first few cleavage faces; only the protein yolk appears to be relatively immobile and unable to pass through the cell walls.

There must therefore be some general condition which controls the disposition of the various regions of egg cytoplasm. Both Raven, and SPIRALLY CLEAVING EGGS 103

Lehmann (1948a,), who has described very similar phenomena in the oligochaete Tubifex, believe that this control is exerted by the cortex. They suggest that the polar regions of the cortex are not disturbed by centrifugation, and that they exert specific attractions on the materials which should form the appropriate pole plasms, so that these cytoplasmic localisations can become reconstituted. This suggestion would tend to suggest similarities between the mosaic spiral-cleaving eggs and the typical regulation eggs of the echinoderms, in which we have seen that the cortex is probably the seat of the epigenetic gradients which play the main part in early development in those forms. It might be, indeed, that in the spiral eggs also the difference between the animal and vegetative cortical regions is graded, as it is in the echinoderms, but that in the former this cortical gradient is very rapidly converted into a qualitative distinction by the attraction of definitely different materials to form the two pole plasms.

Although an attraction between cortical regions and particular types of cytoplasm may be of major importance in controlling localisation in mosaic eggs, it seems doubtful whether it can be the whole story. The restoration of normality in a centrifuged egg is not merely a matter of attracting one specific substance to each end of the original axis, but of redistributing the whole set of substances which have been deranged. Costello (1948), another recent worker in this field, argues that there must be a more generally pervasive system of relations which orders the egg cytoplasm throughout its mass. He refers to this as ‘ooplasmic segregation’ and has advanced an ingenious hypothesis as to its nature, based on the idea of diffusion gradients. It seems not impossible that his ideas and those of Raven and Lehmann will eventually come together; perhaps the polar cortical regions establish the initial difference between the two ends of the main axis, and this is transmitted through the mass of the egg by some diffusion mechanism similar to that which Costello has indicated.

We have already seen that the various regions which become localised in so-called mosaic eggs are not quite so rigidly determined in their developmental fate as the word ‘mosaic’ originally implied. It is also important to realise that in these eggs other mechanisms are at work, which are quite different from the localisation of distinct types of cytoplasm. Raven (1952) has made a particular study of them in Limnea. He found that treatment with lithium, at stages earlier than the twenty-four cell, caused a reduction in the head. The medio-dorsal part was most strongly affected, and the effects show a series of grades leading to complete disappearance of this region and fusion of the eyes. This is a typical example of the type of behaviour spoken of as a ‘field’ process; that is


to say, the whole region is behaving as in some sense a unit within which some property, which controls future development, is distributed in a graded manner. When the region is affected by some substance toxic to it (lithium in this instance) it reacts as a whole, every part as it were sinking in grade, so that the more central parts take on the character normally proper to more peripheral regions.

Again in Limnea, Raven has demonstrated the importance of typical processes of induction. Normally a shell-gland is formed by the ectoderm, in the area where the gut comes into intimate contact with it. Raven was able to induce the blastula to exogastrulate, and showed that if the gut did not touch the ectoderm, no shell-gland was formed. Moreover, in cases of abnormal gastrulation, in which the gut had reached an atypical part of the ectoderm, a shell-gland was formed in this unusual position. Thus there is little doubt that the shell-gland is induced by contact between the gut and the ectoderm. We shall see that such processes play a major role in the development of vertebrates. In the mosaic eggs, although there are probably many more of them than have yet been discovered, they seem to be of secondary importance in comparison with the process of cytoplasm localisation (Fig. 6.5).

FIGURE 6.5 Induction of the shell gland by the archenteron in Limnea.

Normally the shell gland (S.G.) lies posterior to the main band of cilia

(Prototroch, Pr.), where the archenteron comes in contact with the ecto derm (1). But if, owing to abnormal gastrulation, the archenteron reaches

forward to the pre-trochal ectoderm, the shell gland is formed in the corresponding place, (2). (After Raven 1952.)


Hérstadius 1937, Raven 1948, Lehmann 19484, Costello 1948.