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Chapter V Echinoderms
1. Normal development
The early stages in the development of echinoderm eggs are very simple, and provide classical examples of the two fundamental forms, the blastula and gastrula. Moreover, the physiology of these stages has been rather fully investigated. There is therefore much of interest in echinoderm development even for a general account of embryological principles, in spite of the fact that the later stages are highly complex. The simple, early stages of development lead to the formation, not of the adult, but of a larva, which is usually the so-called ‘pluteus’. The insertion of a larval stage into the life-history is, of course, very common among invertebrates. Such larvae fulfil many functions; they may, for instance, facilitate the dispersal of the species, as in many groups of parasites; or they may seem to have been evolved as the quickest way in which the egg can be converted into an animal capable of feeding itself. The latter would appear to be the raison d’étre of the Pluteus; it is a little animal which can swim by means of cilia, and feed itself from the minute life among the plankton. It is only after a considerable independent existence that it becomes converted, by a complicated metamorphosis, into the adult. In this discussion, we shall not attempt to deal with anything more than the first steps of development, by which the larva is produced (Reviews: Lehmann 1945, Horstadius 1939, 1949).
The cleavage of the echinoderm egg is total, and radially symmetrical. The first two cleavages are vertical, through the animal pole. The third is horizontal, running slightly above the equator, so that the upper four cells are rather smaller than the lower four. From this point onwards, the cleavages in the animal and vegetative halves take different courses. At the fourth cleavage, the animal cells divide into a flat ring of eight, while the vegetative ones cleave very unequally into two rings, four large ‘macromeres’ above and four tiny ‘micromeres’ below; the cleavage planes are thus nearly at right angles in the animal and vegetative halves. The same is true at the next cleavage, but here it is in the animal half that the division is horizontal, while in the vegetative half it is more or less vertical. At the sixth cleavage, all the division planes are horizontal, and thus we come to a stage with four rings of animal cells, two rings of macromeres, and two of micromeres, there being eight cells in each ring.
For our present discussion, it is not worth while tracing the cleavage in detail beyond this point (Fig. s.r).
Figure 5.1. The development of an echinoderm. Figure a-f show the early cleavages stages. The shading indicates the levels which have been used in grafting experiments; white is an-1, fine dots an-2, coarse dots veg-1, circles veg-2, and black the micromeres. Figure g shows a section at the blastula stage, at which timie cilia develop on the surface; h, beginning of gastrulation and formation of apical tuft of long cilia; i, the micromeres come free into the blastocoel to form the primary mesoderm p.m.; j, appearance of primitive gut, with blastopore b/; from the tip of this secondary mesoderm (s.m.) is given off, the general ciliation is not shown in this or later drawings; k, horizontal section through same stage as j; I, flattening of ventral side, the skeleton begins to be laid down by the primary mesoderm; m, n, views of young pluteus larva from the original vegetative pole and from the side, showing the development of ‘arms’ from the corners of the ventral surface, and the junction of the tip of the primitive gut with the ventral ectoderm to form the mouth. The dotted line in n shows how the original animal vegetative axis has become bent. (After HG6rstadius 1939.)
During the early cleavages, a space is gradually formed between the cells in the centre of the mass. This enlarges as the cleavage progresses, and fairly soon after the last stage mentioned above, all the cells cohere together to form a smooth hollow spherical ball with this space in the centre. This ball is the blastula, and the central space is the blastocoel or blastula cavity. The simple spherical shape is the most ‘typical’ form the blastula can take; all the many variations which we shall find in other groups can be regarded as modifications of it. In the echinoderms there are few special features to notice; but one may remark that the surface of the blastula soon becomes ciliated, and the tiny embryo escapes from its membranes, and begins an independent life soon after the cilia develop. After a short time, it grows a bunch of special long cilia (the apical tuft) at its animal pole.
Soon after the apical tuft forms, the blastula loses its strictly spherical shape and begins to flatten at the vegetative pole, diametrically opposite the tuft. This is the first sign of gastrulation, which, in essentials, consists in the folding inwards of the vegetative part of the blastula to form a pocket pressed into the cavity of the blastocoel. This pocket is the first rudiment of the gut, and is usually known as the ‘primitive gut’ or archenteron. Its walls make up the endoderm, the innermost of the three fundamental layers out of which the embryo is built. The opening by which the gut communicates with the exterior is the blastopore. Meanwhile, the rest of the surface of the blastula, which is not pressed inwards, forms the outermost layer or ectoderm. Between these, as we have seen, there should be a third or middle layer, the mesoderm. The main way in which the carly development of the echinoderm differs from the general scheme which applies to vertebrates is that here the mesoderm originally lies, not between the ectoderm and endoderm, but right at the vegetative pole. It is formed from the micromeres and the material just above them.
The most vegetative cells begin to break loose separately into the blastocoel even before there has been much infolding of endoderm. These are the ‘primary mesoderm’ cells, and they soon start secreting the first calcareous spicules of the skeleton. A similar process of shedding cells into the blastocoel goes on particularly from the tip of the primitive gut as it folds in; this is the ‘secondary mesoderm’, which later forms the greater part of the skeleton and muscles.
The pocket of endoderm pushed in to the blastocoel continues to elongate, producing a long finger-shaped primitive gut. The process is known as ‘invagination’, a term which is applied to all types of movement by which the endoderm and mesoderm are formed from the blastula; as we shall see, it covers several different sorts of foldings and cell migrations. In the echinoderms, it appears to be a simple in-pushing, like that produced when one presses a thumb into a soft hollow ball. While this is going on, the first signs of bilateral symmetry appear. The embryo, now entitled to the name of gastrula, has become somewhat conical. It begins to flatten on one side, which is the future ventral side. The elongating primitive gut turns towards the upper end of this side, fuses with it, and eventually breaks through to form an opening. This is the rudiment of the mouth; and there isnow a complete tube leading from it to the blastopore, which from now on functions as the anus. The gut soon begins to differentiate into an oesophagus, a stomach and an intestine; and meanwhile four long arms grow out from the corners of the flattened ventral side, while the opposite corner of the gastrula also elongates into a single thick spike. This completes the formation of the pluteus.
2. The gradient system
Echinoderm eggs were some of the first with which experimentalists tried to solve the fundamental physiological problems of development. They are easily obtained in large numbers; and it is simple to free them of their membranes (by squirting them through a narrow pipette, for example). Only their small size is an impediment; but this, so long a major limitation on the kinds of experiment possible, was largely mastered by the Swedish investigators Runnstrém, Horstadius and Lindahl, who combined subtle chemical methods of attack with the most delicate manipulative skill.
In the earliest experiments, which were mentioned on p. 62, Driesch showed that if the first two, or first four, blastomeres are separated by cutting along the cleavage planes, each isolated cell can form a complete well-proportioned pluteus. He concluded that these cleavages do not separate parts whose developmental fates have been already determined; the later developmental history of a blastomere can be altered if the cell finds itself in an abnormal situation, as it does when isolated. This conclusion still stands. The cleavage pattern can be altered by various treatments, and in spite of this, normal larvae are formed. Clearly the pattern of cleavage has no decisive effect on the pattern of development. .
Driesch went further in his conclusions and considered that every part of the early egg had the same potentialities. He argued that the formation of a complicated embryo from an egg all of whose parts were alike was an inconceivable achievement to be accomplished by a natural mechanism, and that its explanation demanded a non-natural agent, whose functions were to create order out of uniformity; this he called ‘the entelechy’. But as a matter of fact, Driesch’s basic postulate, that all parts of the egg are similar, has turned out to be untrue; and the entelechy thereby loses its main support.
The demonstration of differences within the egg has come from experiments in which the parts have been cut apart along horizonal planes. There is no need here to attempt to summarise the whole massive volume of evidence, and we will select one of the most demonstrative experiments—that in which the 32- or 64-cell stage has been dissected. Hérstadius, who did the experiment, distinguished five zones. Animal-1 and animal-2 lie at the top, and below them are vegetative-1 and vegetative-2 derived from the macromeres; at the very bottom of the egg there are the micromeres. In the first part of the experiment, each of these zones was isolated. The essential feature of the result was that each zone developed more or less in accordance with its normal fate within the embryo, only more so, if one may put it like that. For instance, an-1 gave a pluteus which contained only the organs appropriate to the most animal region (c.g. apical tuft, no gut); but these organs were exaggerated, in the sense that the apical tuft spread out to cover nearly the whole surface. An-2 also gave larvae with no gut, and usually with apical tufts, which were often enlarged (although normally an-2 does not participate in the formation of the tuft, which is formed from an-t cells). Similarly the vegetative cells gave larvae containing vegetative organs, which again might be exaggerated. The micromeres unfortunately fail to develop anything when isollated, but veg-2 gives a larva with an exaggerated gut, which is often too large to fit inside the ectoderm and thus protrudes, the resulting embryo being known as an exogastrula. Veg-1 is highly variable; sometimes it has exaggerated vegetative organs (e.g. gut), sometimes it gives a larva consisting mainly of animal organs; in a few cases it produces a reasonably normal pluteus.
Combinations of the animal half with various levels along the animalvegetative axis: A, the isolated animal half (an-1 plus an-2) forms a blastula nearly completely covered with apical cilia, and later a larva with no gut; B, the addition of veg-1 restricts the size of the apical tuft, but again no gut develops; C, with veg-2 a relatively normal embryo is formed; D, with veg-1 plus the micromeres, the embryo forms a gut which is abnormally large; E, the addition of the four micromeres to the animal half gives a near ly normal larva, although their mass is much smaller than the veg-2 material added in C. (After Horstadius 1939.)
Hérstadius interpreted these results to mean that the egg contains two gradients, an animal one with its high point at the animal pole, and a vegetative one running in the opposite direction. Regions which are high on the animal gradient tend to form animal organs, and similarly for the vegetative. Moreover, Hérstadius proceeded to show that the two gradients interact with one another. In a very beautiful series of experiments, he combined the various layers in abnormal combinations. He found, for example, that normal plutei were formed fairly frequently when an-1 was combined with four micromeres, and that as the number of micromeres was reduced, so more and more ‘animal’ larvae appeared. An-2, on the other hand, required only two micromeres to produce a normal larva, and became vegetative in character if more were added. Veg-1 was usually swung over too much to the vegetative side by the addition of even one micromere; probably about a half would be enough to counteract its slight preponderance of animal tendencies (Cf. Fig. 5.2).
Both in the experiments in which the various zones were isolated, and in those in which micromeres were grafted, there was considerable variation in the resulting embryos. It appears that one is dealing not only with the interaction of animal and vegetative potentialities of various strengths, but that there is also another important factor, namely a tendency for the normal equilibrium between these two conditions to be restored, even when the two parts originally grafted together were not in perfect balance (see Lehmann 1945, p. 64). We will find that a similar tendency to restore a normal equilibrium condition is a very frequent influence in most developmental events. It is an aspect of individuation (p. 12).
There are several other types of experiments in which individuation is strikingly exhibited in the echinoderm embryo. The vegetative region, and particularly the micromeres, have a very strong ability to influence their surroundings in such a way that the latter fit into the building-up of a complete or partially complete embryo. For instance, a meridional half may be combined with an animal half in the 16-cell stage (Fig. 5.3). From this a normal pluteus will develop, the part of the animal material lying next the micromeres being, as it were, absorbed into the developmental system of the latter and converted into gut. A rather similar result is obtained if a group of micromeres is inserted among the animal cells of an intact egg (Fig. 5.4). They continue developing in a vegetative way, and, further, swing some of the neighbouring animal material into line with them, so that it too develops as gut. The animal gradient of the egg is exhibited by the fact that the nearer the animal pole the graft is placed, the smaller is the gut formed.
It is important to note the similarities, and also the differences, between the production of a gut by the action of the grafted micromeres on the animal cells in the above experiments, and the ‘embryonic induction’ which is most strikingly seen in vertebrates. In both cases, a part of the embryo, when grafted into an abnormal situation, causes the material surrounding it to develop in a way which it would not normally have done. In true embryonic induction, however, the graft induces from its surroundings something of a different nature to itself; for instance, in the vertebrates, mesoderm can induce the formation of neural tissue, or in Limnea the gut can induce the formation of the shell-gland. In the echinoderms, the micromeres induce something which is either normally derived from micromeres or from the next most vegetative material; we are dealing with a tendency for the completion of the graft at the expense of its surroundings; moreover, these surroundings clearly play an important part in determining the nature of the structures induced. In the vertebrates, as we shall see, induction may sometimes show certain features which indicate a similar assimilative or individualising tendency, but it can also be a much more independent process, the inducer provoking the appearance of something which it does not in any way need in order to complete itself. Thus vertebrate induction may involve both individuation and something else, which has been named ‘evocation’; in the early stages of the echinoderm, we seem to have only the former.
Figure 5.3. A meridional half of the 16-cell stage (uncoloured, on left, containing the whole an-veg axis) is combined with the animal half of the same stage (shaded). A normal embryo develops, shown in section on the right. (After Horstadius.)
The hypothesis of two gradients finds very strong support in the work described above. But innumerable questions immediately suggest themselves. What are these things gradients of? Are they localised in any particular part or structure of the egg? And how do they produce their effects? None of these questions can be answered with any assurance, but knowledge is increasing rapidly about them. As regards the first, we know that the gradients can be influenced by chemical substances (Lindahl 1942, Gustafson 1950). The vegetative tendencies are strengthened by the action of lithium salts. If complete eggs are treated with lithium, they form too large a gut, producing exogastrulae similar to those formed when veg-2 is isolated. Similarly, isolated animal halves can be brought to develop into normal larvae by lithium. The animal tendencies are increased by early treatments with thiocyanide. This, and other similar evidence, suggests that the gradients are fundamentally based on the rates of certain critical chemical reactions. Moreover, if young embryos are constricted, by being tied in a loop of hair, the degree to which the animal and vegetative gradients interact with one another depends on the width of the connection between the two halves, which again suggests that diffusing chemical substances are involved.
Figure 5.4. Grafts of the micromeres (black) at different levels along the an-veg axis. They migrate into the blastocoel cavity as primary mesoderm, and induce the formation of an archenteron from the material near them. This induced gut (indicated in black in the right-hand column) is larger the nearer the graft is to the vegetative pole. (After Hérstadius.)
We still have little idea what these substances are. Very careful measurements have failed to reveal any difference in respiration between the animal and vegetable halves. However, Child (1936) observed some years ago that by the blastula stage there is a double gradient in the rate of reduction of vital dyes such as Janus Green and methylene blue, which are indicators of redox-potential. There is a high level of reduction activity at the vegetative pole, falling off towards the animal, and simultancously a weaker gradient running in the opposite direction from the animal pole towards the vegetative. Hérstadius (1952) has recently studied these gradients in isolated animal and vegetative halves, and in animal halves into which micromeres have been implanted, and has shown that their behaviour parallels that of the postulated gradients of animalvegetative tendencies. The biochemical meaning of the gradients in dye reduction is not yet understood, nor can one be certain whether they are related to the causes of the animal and vegetative gradients or are merely among their effects (Fig. 5.5).
A perhaps more promising clue to the processes underlying the gradients is the fact that in rather late cleavage stages the vegetative region of the egg comes to require the presence of sulphate ions in the medium, and its development is inhibited in artificial seawaters from which they are absent. Lindahl (1942) suggests, on these and other grounds, that the reactions proceeding in the vegetative region give rise to aromatic waste products (formed from protein catabolism) which are normally disposed of by being combined with sulphate. Hérstadius and Gustafson (1954) have studied the animalising and vegetativising effects of various aminoacids as well as substances which might be expected to play a role in carbohydrate metabolism. The latter group were, on the whole, animalising, which supports the suggestion that the animal tendencies are connected with carbohydrate metabolism; but the effect of the amino-acids, although usually towards vegetativisation, was not invariably so, and further work will be necessary before the experiments can be fully interpreted.
Meanwhile there is another quite different theory in the field. Ranzi (1951) presents evidence to show that lithium and thiocyanate have important effects on the viscosity of solutions containing elongated protein molecules, and he believes that their actions on the echinoderm egg are related rather to their influence on the physical condition of the cytoplasm than to any direct alteration of the chemical metabolism.
Finally, a very unexpected point has recently been discovered by Lindahl (1953) who has claimed that, in the two species of sea-urchin he studied, the micromeres are haploid in chromosome number. It is not clear how this remarkable example of nuclear differentiation (cf. p. 354) is related to the high ‘vegetative’ activity of these cells.
As regards the localisation of the gradients, most authors agree that they must be in the cortex. The argument is primarily the negative one, that the internal regions of the egg can be shifted about by centrifugation without producing any profound result, so that the controlling gradients must be in the stiff ectoplasmic layer which is not displaced by centrifugation.
Figure 5.5. Sections of early gastrulae stained with Janus Green, which is at first blue (large dots) changing to red (fine dots) as it becomes reduced; in the undotted areas the colour has faded altogether. A 1, 2, 3, three stages over a period of 50 mins. in an isolated vegetal half, showing the reduction spreading from the most vegetative region. B 1, 2, 3, a period of 55 mins. in an isolated animal half, the reduction starting in the most animal region. C, an animal half into which micromeres have been grafted, showing the induction of an archenteron, with an associated region of rapid reduction, and the slow progress of reduction in the neighbourhood of the apical tuft. D, micromeres were implanted on the left between an-1 and an-2 of a normal 32~cell stage, as in Figure 5.4 middle row; a region of rapid reduction has appeared in their neighbourhood. (From Hérstadius 1952.)
Finally, to the question of how the gradients work, we can offer at least the beginning of an answer. Although moderate centrifugation, sufficiently strong to cause considerable stratification of the cytoplasm, has no great effect on development, the situation is rather different following very intense centrifugation in a modern ultra-centrifuge. Using forces of the order of 45,000 g, on the unfertilised egg, Pease (1939) was able to throw down to the centripetal end a mass of small mitochondria-like granules. He found that if, owing to the orientation of the egg in centrifuge, these had been collected at the animal pole, the vegetative pole which lacked them was unable to gastrulate; and so, mutatis mutandis, was the animal pole unable to develop if all the granules had been forced to the vegetative end. He suggested that these granules are the immediate agents of differentiation.
At the present time, a great deal of attention is being paid to the behaviour of various types of cell granules in developing echinoderms. They can be roughly classified into ‘mitochondria’, which are larger and become sedimented at relatively low speeds of the centrifuge (giving about 16,000 ¢g), and ‘microsomes’, which are only sedimented at the highest speeds (giving about 100,000 g). The mitochondria can be detected by normal cytological techniques within the cells of the embryo. It has been found (Gustafson and Lenique 1952, Lenique, Hérstadius and Gustafson 1953) that in early stages they are distributed in a gradient, decreasing in concentration from the animal to the vegetable pole (Fig. 5.6). In isolated animal or vegetative halves, and in halves which have been ‘animalised’ or ‘vegetalised’, the gradients become altered in a manner exactly parallel to that of the basic animal and vegetative gradients, or the gradients in dye reduction mentioned above. This makes it most probable, then, that the mitochondria are rather directly concerned with the fundamental developmental processes. Gustafson (1953, 1954) has made extensive studies on their metabolism, and suggests that they are connected with the synthesis of the fibrous proteins of the apical tuft, which is the most characteristic animal organ.
The mitochondria can be formed anew during development. Harvey (1946) centrifuged echinoderm eggs in a medium which had a gradient of specific gravity, and showed that under these circumstances, the egg may be split into several fragments, each containing the constituents of a particular density. Fragments made in this way may develop normally even if they originally contain no mitochondria; but it is found that mitochondria gradually appear. It seems probable that they are formed from the cytoplasmic particles of smaller size (the microsomes). Hultin (19534, b) has summarised a number of studies in which radioactive isotopes have been used to follow the processes of protein synthesis in the cell as a whole and in the various groups of particles (see also Kavanau 1953). During the early cleavage stages, it is in the microsomes that this synthesis is proceeding most rapidly, but by the early gastrula the mitochondria are becoming very active. Hultin suggests that the mitochondria are built up either from, or at least by the influence of, the microsomes. The reactions between these two types of particles are, however, probably reciprocal, since there is reason to believe that, once the mitochondria have been formed, they induce a high rate of activity in the microsomes, and that it is actually at these small particles that protein synthesis proceeds most rapidly. The evidence from work with isotopes suggests that this synthesis begins to get fully under way at about the time of gastrulation.
Figure 5.6. Graphs showing the relative density of the mitochondrial population (R.M.D., plotted vertically) at various levels of the animal-vegetative axis at 7, 14 and 26 hours of development (blastula to mid-gastrula). (From Gustafson 1954, after Lenicque, Hérstadius and Gustafson 1953.)
Perlmann (1953) has indeed been able to detect, immunologically, the appearance of at least one new antigenic substance (protein?) at that time.
The study of the roles of the various types of cytoplasmic particle has only begun very recently and there is still much to do. Hultin emphasises a point which is probably of considerable general importance, namely that we must think of the developing cell as containing populations of mitochondria and microsomes which are continually reacting on and influencing each other; and one may add, of course, that the nucleus with its contained genes must be also involved in the same general network of cause and effect.
All the grafting experiments on the gradient system described above were carried out on the early cleavage stages, before the embryo has sixty-four cells. Isolations of animal and vegetative halves can be made at later stages, and it is found that the divergence of the isolate from its normal fate remains much the same until the early blastula stage (about ten hours after fertilisation). From that time onwards, the developmental fate of the regions rapidly becomes more fixed, so that, for instance, an animal half isolated from a later stage develops only the normal sized tuft of cilia, and a vegetative half only a normal gut. The developmental fate is not entirely determined in such halves, since if micromeres are implanted into animal halves isolated at various times, it is found that they can induce some vegetativisation even in halves which, if left isolated, would develop only into their original presumptive fate. The embryonic materials are, however, rapidly losing their lability, and by about sixteen hours after fertilisation, an animal half can no longer be affected by implanted micromeres. It is interesting to note that if an animal half is isolated at the 32-cell stage, and allowed to develop in isolation for some time before the micromeres are grafted into it, it loses its reactivity earlier than it would do as part of a whole egg; presumably its isolation from its vegetative partner allows its animal tendencies to become fixed earlier.
3. The dorso-ventral axis
It was stated earlier (p. 48) that the determination of a dorso-ventral axis, which confers a bilateral symmetry on the egg, is one of the fundamental steps in development. It may well be asked how, and at what time, this step is taken in echinoderm development. As a matter of fact, the evidence suggests that there is some trace of bilaterality even in the unfertilised egg, presumably dependent on factors operating during the maturation of the egg in the ovary. If such eggs, or early cleavage stages, are sectioned vertically, there are slight differences in development according to the plane of the section. These can be best interpreted by the hypothesis that one side already has a slight tendency to become ventral, and usually does so. If a ventral half is separated from a dorsal half, the ventral face appears in its original position in the former, but in the dorsal half, the axis may be reversed, so that the eventual ventral side of this halfembryo appears on the side which, in the undisturbed egg, would have been dorsal.
This original organisation is only a labile one, and can be overcome by a number of different influences. It becomes determined at about eight hours after fertilisation. If eggs are strongly stretched before this, by being sucked into a tube with a narrow lumen, the dorso-ventral axis is altered so as to run along the length of the elongated egg. Strong staining of one end of such an egg with Nile Blue sulphate will cause this end to become dorsal; perhaps it would be better to say that it will cause the other end to become ventral, since it is probable that the ventral side plays the lead in the development of the axis, and that it is a suppressive action of over-staining which is the operative mechanism in the experiment. Many other chemical substances can influence the determination of the axis (see Review in Lehmann 1945). Gustafson and Savhagen (1949) have recently shown that weak solutions of detergents will suppress the development of the oral or ventral side entirely, so that radially symmetrical larvae are formed. A similar result was produced by Horstadius and Gustafson (1954) who treated the eggs shortly after fertilisation with antagonistic analogues (or ‘anti-metabolites’) of certain vitamins, etc. They suggested indeed that these substances were actually operating as detergents rather than by inhibiting the growth-promoting properties of the vitamins.
Hérstadius 1939, 1949, Gustafson and Lenique 1952 or Gustafson 1953, 1954, Hultin 1953).
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