Waddington1956 7
Embryology - 19 Apr 2024    Expand to Translate
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Waddington CH. Principles of Embryology (1956) The MacMillan Co., New York
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Chapter VII The Ascidians and Amphioxus
The ascidians stand midway between the invertebrate and vertebrate kingdoms. In the early development of many of them, a tadpole-like larva is formed, which is furnished with an axial notochord with an accompanying dorsal nerve-cord. These formations disappear or become greatly modified in the final metamorphosed adult, but in so far as the animal possesses them at all, it is to that extent entitled to be reckoned as a member of the chordates. It is more doubtful whether the ascidians should be regarded as exceedingly primitive members of the group in which the larva foreshadows the later evolutionary history, or as a specialised group of degenerate forms in which the chordate larva is the only remaining sign of a more glorious evolutionary past. Perhaps both views have something of the truth; the ascidians may represent a chordate stock which began to degenerate after only a short history of progressive evolution. In favour of this interpretation is the fact that in the very early development of the egg, which leads to the formation of the chordate larvae, they show many of the features which one would expect in a primitive, as opposed to a highly evolved, member of the Chordata. The eggs, in fact, bear a very striking resemblance to those of Amphioxus, which is undoubtedly a very primitive chordate. It will, indeed, be convenient to treat the early development of ascidians and Amphioxus together. The simplicity and clarity of their developmental processes, together with the primitive position of Amphioxus in the evolutionary scheme, has for long rendered these animals classical embryological material.
The first full study of the Amphioxus egg was made by Hatschek in 1881. Unfortunately many of the authors who followed him (Wilson, MacBride, Cerfontaine) misinterpreted the orientation of the early embryo, thinking that the anterior was the posterior and vice versa; and some textbooks still follow their erroneous accounts. The true state of affairs was made out by the American embryologist Conklin (1932). In the earlier years of the century, the same author had laid the foundations of our knowledge of ascidian experimental embryology (Conklin 1905).
In both Amphioxus and the ascidians, the egg is fairly small (about 0-1 to o'2 mm. in diameter) and contains only a moderate amount of yolk. Before fertilisation, the egg nucleus is in the form of a large germinal vesicle, with a diameter up to half that of the egg. It lies at one side, closely against a peripheral layer of clear cytoplasm which encloses the main bulk of the egg. Conklin points out that in Amphioxus, and probably in ascidians, the attachment of the egg to the wall of the ovary is by the end containing the germinal vesicle, that is by the animal pole, whereas in most invertebrates it is the vegetative pole which is attached; this difference he correlates with the fact that the vertebrates have a dorsal nerve cord and the invertebrates a ventral one, so that the two kingdoms seem to differ by a reversal of their dorso-ventral axis. The suggestion is an interesting one, but it is by no means clear that most vertebrate eggs are like Amphioxus in having their animal pole attached to the ovary wall. In the frog, the point of attachment seems to be slightly below the equator.
Both ascidians and Amphioxus provide beautiful examples of the important fact that an egg is not a mere lump of featureless cytoplasm furnished with a haploid nucleus, but on the contrary has an effective architecture of its own. The eggs of some ascidians, such as Styela, described by Conklin, are perhaps the clearest instances of this to be found in the animal kingdom, since in this form several different regions of cytoplasm can be visibly distinguished owing to their content of coloured granules, mitochondria, yolk, etc. Before fertilisation, the egg of Styela has a peripheral layer of clear yellowish cytoplasm, inside which is a grey yolk-laden cytoplasm, and the large germinal vesicle filled with clear sap (Fig. 7.1). The sperm enters always near the vegetative pole. As it penetrates it sets off a reaction of the egg surface, which in this instance involves a streaming of the yellow peripheral cytoplasm downwards to the vegetative pole. Simultaneously the germinal vesicle undergoes the reduction divisions and breaks down to give rise to the polar bodies; the clear plasm thus released also moves down the egg and lies above the yellow material just above the vegetative region. The sperm head now starts to move upwards, remaining fairly near the egg surface. As it does so, it appears to pull the two layers of clear and yellowish cytoplasm along with it, the yellowish material remaining on the surface, while the colourless extends deep into the centre of the egg. After throwing off the polar bodies, the egg nucleus descends towards the sperm nucleus, and they meet just below the equator but away to the side of the egg towards which the sperm has been travelling. Since the yellow and colourless materials have been accompanying the sperm, they are now seen as two crescents, clear above and yellow below, with their thickest part in the meridian along which the sperm moved. This meridian is clearly a plane of bilateral symmetry in the egg. The first cleavage plane falls along it, and in the final embryo it is the plane dividing the right side from the left. The region where the two nuclei fuse, and where the two crescents are thickest, is in the eventual posterior and ventral part of the embryo, while the opposite side of the egg is the anterior-dorsal.
Shortly after fertilisation, the clear cytoplasm spreads over the greater part of the animal half of the egg. The original central material of the unfertilised egg, the grey yolky plasm, is now confined to the vegetative region. It gradually becomes differentiated into a darker mass at the vegetative pole, and a lighter grey material which forms a crescent at the opposite side of the egg to the yellowish stuff. The egg thus comes to have a fairly complicated architecture.
These various regions of the egg are highly significant for the future development. By careful observation, Conklin could follow each region through the subsequent stages, and determine which organs it eventually formed. The end-product which a given region of the egg (or early embryo) will eventually form if left to itself in the intact egg is spoken of as its ‘prospective fate’ (sometimes the expression ‘presumptive fate’, derived from the German, is used). Conklin was thus in a position to make a map of the early cleavage stages, marking on it the prospective fate of the various parts; in fact, in Styela, the map was more or less made for him by the colouration of the various regions. This is not usually the case. In Amphioxus, for example, although the general set-up is probably
almost identical with that in Styela, only the peripheral cytoplasm can be distinguished (although it corresponds with the yellowish material in Styela it is grey in Amphioxus). In most other organisms there is no overt sign which distinguishes the regions of different prospective fate, and much ingenuity has had to be used to keep track of the parts of the early egg and discover where they get to and what they develop into. In spite of the technical difficulty of obtaining them, prospective fate maps are the clearest way of summarising the future course of development, and we shall use them again and again to show how the embryological events which look so different in different groups of vertebrates, can really be traced back to the same general scheme.
File:Waddington1956 fig7.1.jpg
Figure 7.1. The development of the Ascidian Styela. (a) Before fertilisation, showing the large germinal vesicle(g.v.) and the peripheral yellowish layer (y), and the inner grey yolky cytoplasm. (b) Immediately after fertilisation. The germinal vesicle has ruptured and released a clear grey cytoplasm. The yellow cytoplasm is accumulating at the vegetative pole. (c) The sperm nucleus s is visible in the yellow cytoplasm which lies at the vegetative pole. The clear grey cytoplasm has moved down and lies just above the yellow. (d) Shortly before the first cleavage. The sperm nucleus has moved up and met the female nucleus just below the equator. The yellow and grey cytoplasms have moved up with the sperm nucleus. The yellow crescent is more or less superficial, the grey one extends into the depth of the egg. (e) Two-cell stage. The grey crescent is becoming more diffuse. (f) Eight-cell stage from side. (g) Sixteen-cell stage from animal pole. Note the bilateral symmetry. (h) Early gastrula from vegetative pole, looking into the wide open blastopore b; on the dorsal side of this are the future neural cells #.p and chordal cells c. (i) Longitudinal section through young larva. n.p. neural plate, cnotochord, e ectoderm, end endoderm lining the archenteron, m mesoderm, b blastopore. (In this and Figures f, g and h the yellow crescent material is shaded so that its movements can be followed but it cannot actually be recognised by its colour so clearly as the diagram suggests.) (j) Longitudinal section through a young larva: ¢., notochord; ey., eye spot; end., endoderm; t.e., endoderm of tail; mes., mesenchyme; #., nerve cord; n.v. neural vesicle; pap., adhesive papilla. (After Conklin 1905.)
File:Waddington1956 fig7.2.jpg
Figure 7.2. Presumptive areas in the 8-cell stage of an ascidian, seen from the right side. White, ectoderm; a, mesoderm (muscle); b, endoderm; c, notochord; 1, spinal cord; tg, brain. (After Vandebroek 1938, Reverberi 1948 and Ortolani.)
The prospective fate map of the ascidians, and of Amphioxus which is essentially similar, is shown diagrammatically in Fig. 7.2. It has recently been carefully studied by Vanderbroek (see Reverberi 1948) and Ortolani (1954). It will be seen that the yellow crescent of Styela, which corresponds to the grey crescent which alone is distinguishable in Amphioxus, eventually becomes mesoderm. It is thus not the constituent which forms the main body axis, since that is composed of the neural plate with its underlying chorda. The original locations of these two organs are found on the other side of the egg, in the light grey area of Styela, which does not appear distinctly till after fertilisation. We shall see (p. 146) that in Amphibia it is the material for the chorda, more or less corresponding to this light-grey crescent, which first becomes visibly distinguishable; it is important to remember this difference between the two forms, which we shall find are otherwise very similar in their general pattern. Above the ring formed by the mesodermal and chorda-neural crescents lies an area which will form ectoderm, and which in Styela can be seen to originate in the clear cytoplasm which emerged from the ruptured germinal vesicle; below lies the dark-grey yolky material which develops into endoderm.
It is now necessary to trace the movements and changes by which the prospective areas attain their fate. These are perhaps slightly clearer in Amphioxus (Fig. 7.3), but essentially the same features can also be seen in the ascidians. The first cleavage plane, as has been said, lies in the plane of bilateral symmetry which bisects the mesodermal and chorda-neural crescents, and runs through the animal and vegetative poles. The second plane is also vertical and is at right angles to the first, cutting off two slightly smaller cells at the posterior side, and two larger ones in front. (It was in regard to the orientation of this and the later stages that the earlier workers were mistaken.) The next cleavage is horizontal, giving an 8-cell stage in which the lower group of cells are all slightly larger than the corresponding upper ones. Further than this it is unnecessary to follow the cleavages in detail. As they proceed, a jelly-like material accumulates in the centre of the mass of cells, which are gradually pushed outwards to form a hollow sphere, which is a blastula of the typical form we have already seen in the echinoderms.
The gastrulation process, by which this blastula becomes converted into a three-layered embryo, begins by a slight flattening of the vegetative end. The cells in this region are those derived from the dark-grey yolky material and are somewhat larger than any others in the blastula, so that they form a rather solid-looking coherent flat plate. This sinks into the interior of the hollow blastula. The opening which leads in from the exterior towards the sunken plate is the blastopore, its edges the blastoporal lips. The cavity into which it leads, which grows deeper as the plate sinks further into the interior, is the primitive gut.
The shape of the blastopore alters somewhat as the gastrulation proceeds, but there is no need to repeat here all the details given by Conklin in his classical account of Amphioxus. At first the endoderm plate is triangular, with a wide straight dorsal lip and two lateral boundaries which converge towards the ventral lip. Although the sinking in of the endoderm begins ventrally, it proceeds fastest on the dorsal side, and it is the dorsal lip which is the most sharply inflected. In Styela this lip is originally made up of about six transverse rows of cells, each row containing rather more than a dozen cells, all of which have been derived from the light-grey chorda-neural crescent. The lower three rows lie inside the lip, and come into the roof of the primitive gut; these give rise to the chorda, while the remaining three rows stay on the outer surface and become the neural plate. During the gastrulation, the arrangement of the cells in the rows is profoundly altered; they slip between one another, so that the chorda and neural plate, whose material was originally arranged in transverse rows, become longitudinal strands running forward from the blastopore lip. The effect of this is that the cells of the mesodermal crescent on the other side of the egg have to move over towards the dorsal side to fill the gap which would otherwise be left; and as the stretching of the dorsal organs continues, the primitive gut becomes a tube with a strip of chorda along its most dorsal part, a strip of mesoderm on each side of that, and the main hollow of the tube lined with endoderm.
As the elongation proceeds, the blastopore narrows like the mouth of a laundry bag when the string is pulled tight. Before it is completely closed in Amphioxus the ectoderm just above the ventral lip grows up in a curved ridge, the sides of which rapidly come together, covering the still-open blastopore from sight, and progress forwards above the neural plate, like two flaps being drawn together by a zip-fastener up the mid-dorsal line. Underneath these flaps, the neural plate continues to get longer and narrower, and its centre sinks down to form a trough or groove; in the most anterior end, indeed, it rolls up completely so as to produce a neural tube, such as we shall find in the higher vertebrates. Meanwhile important changes are beginning in the walls of the archenteron. These can be most simply described by saying that the endoderm, which in a cross section still makes up only the ventral and lateral walls of the gut tube, now starts to grow inwards from each side towards the dorsal midline. In doing so, it first undercuts the mesodermal strips, pushing them outwards as grooves which eventually become completely cut off from the gut as hollow sacks; and after pushing off the mesoderm, it then grows under the chorda, excluding it also from the walls of the gut, which thus becomes completely lined by endoderm. It should be noted that although this is a convenient method of describing what happens, we do not actually know that it is primarily the endoderm which pushes off the mesoderm, and not the latter which actively withdraws itself; such questions could only be definitely answered by an experimental analysis which has not yet been made. It may well be the mesoderm which plays the active part in its removal from the gut wall, since soon after this it undergoes a change for which the endoderm can scarcely be made responsible; the long hollow sacks of mesoderm become nipped off into a series of short, square chambers, which lie in orderly pairs on each side of the chorda; these are the somites.
File:Waddington1956 fig7.3.jpg
Figure 7.3. Development of Amphioxus.
(a) Section through egg one hour after fertilisation, showing the conjugation of the pronuclei, a polar body, and the grey crescent material on the left (posterior) side.
(b) Eight-cell stage, seen from right side.
(c) ae stage. A space is appearing in the middle of the group of cells.
(d) A left half of an early gastrula, seen from the right side. Note large blastocoel cavity, the plate of large endoderm cells on the lower right (anterior) side of the drawing and the irregular, rapidly dividing cells of the mesodermal (former grey) crescent on the lower left.
(e),(f) Similar views of later gastrula stages.
(g) Gastrulation nearly completed and the blastopore narrowed to quite a small hole.
(h) A dorsal view just after the completion of gastrulation. The blastopore is at the bottom, but is covered by the two flaps of ectoderm which are growing up over the neural plate, leaving a pear-shaped area of it visible. The small circles arranged in a figure of eight are the nuclei in the endoderm cells lining the archenteron.
(i) Optical section at a slightly later stage, in the same orientation as g and the previous drawings. Dorsal to the archenteron (arch.) the left row of somites is seen lying above the notochord, and dorsal to that is the neural plate, covered by the flap of ectoderm; blastop =blastopore.
(j) Left view of 48-hour larva: gt. gut; n.t., neural system; ch, notochord. (k) Section through the level of the second somite in Figure i, showing the neural plate (dotted) beneath which are the notochord, and the somites which are folding off from the walls of the archenteron.
(!) Section through same somites somewhat later. The neural plate is folding into a groove; the somites have separated from the gut-wall and from the notochord, and the coelomic cavity has expanded within them. (After Conklin 1932.)
Only the first eight or nine pairs of somites are formed in this way as outpushings from the gut wall. By the time they are laid down, the blastopore, hidden under the ectodermal fold, is nearly closed. The further growth in length of the larva is brought about by the proliferation of all the material lying round this remnant of blastopore. This mass of rapidly dividing cells grows out posteriorly, differentiating directly as it does so into neural plate, notochord, somites and endodermal gut wall. It has been suggested that in higher vertebrates too the more posterior regions of the body are formed from a general proliferating mass in which the embryological processes are by no means so clear-cut as they are in more anterior regions; but we shall see (p. 263) that recent work makes this unlikely.
There is no need for our present purposes to follow the later history of the Amphioxus larva in detail. It should perhaps be mentioned that the mouth forms as an opening which breaks through into the gut near its anterior end, while the anus is a similar opening nearer to the site of the blastopore remnant, but somewhat anterior to it. The somites, from a fairly early stage, expand laterally, growing round between the ectoderm and endoderm till they fuse in the mid-ventral line, and thus provide the gut with a complete mesodermal clothing. These lateral extensions of the somites are, from the first, hollow like the somites from which they come, and the space between their inner and outer walls is the origin of the final body-cavity or coelom. If this cavity is traced back, it will be found to be derived directly from the cavity of the gut or enteron; whence a body-cavity originating in this way has been called an entero-coel, in distinction to the schizo-coel (formed by splitting of an originally solid layer) which is characteristic of most vertebrates. But it seems probable that the distinction is not such a fundamental one as some of the older embryologists thought.
The eggs of ascidians have been favourite material for experimental study for many years; Amphioxus material is less easily come by and much less is known about it, though what data do exist suggest that its epigenetic physiology is not greatly different from that of the better-known group. The older workers found much evidence that the ascidian egg is a typical mosaic type. In particular, Conklin (1931) showed that after fairly strong centrifugation which moved around the interior cytoplasm abnormal larvae develop, their build corresponding to the new positions to which the ooplasms had been shifted. He found, however, that the essential factors which determine the mode of development of the regions are not the inclusions to which the colours of the various crescents are due. For instance, the yellow granules can be moved out of the yellow crescent by mild centrifugation, but the mesoderm still appears in its normal place so long as the hyaline ground-substance has remained in place. Ries (1939, see also Reverberi and Pitotti 1939) later found that the property of developing into mesoderm (and particularly muscles) is bound up with some cytoplasmic constituent which has a high peroxydase activity; possibly these are ultra-microscopical granules. Wherever the peroxydasecontaining material is forced to by the centrifugal force, there the muscles will eventually develop.
In recent years, it has become clear that the location of specific ooplasms in definite regions of the egg is only one part of the story (Review: Reverberi 1948). The first important additional information concerned the mechanism by which the localisation is brought about. Dalcq (1932) found that, if eggs are cut into two parts before being fertilised, two complete larvae can be produced (provided the cut is meridional, so that each fragment contains the whole animal-vegetative axis). This is a typical ‘regulation’, similar to that found in the echinoderms. It certainly indicates that considerable redistribution of the ooplasms can occur in the unfertilised egg. Reverberi (1936) in similar experiments studied the capacity of egg fragments to regulate sufficiently to carry out a normal bilaterally symmetrical cleavage. He found that they retained this degree of flexibility up to the time of the emission of the polar bodies, but that the regulation is dependent on some factor located in the vegetative region, in the absence of which the animal part alone cleaves as though it remained only part of an egg, and does not develop bilateral symmetry. Thus in the egg before and just after fertilisation there is a considerable capacity for regulation, which can be shown to involve mutual interactions between one part and another.
Evidence has also accumulated that even after the ooplasms have become localised, reactions between the parts of the embryo are by no means at an end. The details of Dalcq’s experiments with fragments of unfertilised eggs had already suggested this possibility to him, and conclusive evidence of regulation in the 2-cell stage was found by von Ubisch (1938), who was able to cause two eggs at this stage to fuse together, when in some cases only a single perfectly normal larva was developed.
Much fuller information has been obtained from experiments in which, at the 8-cell stage, the blastomeres have been separated and recombined in various ways (cf. Rose 1939, Reverberi 1948, Reverberi and Minganti 1953). Quite a complicated cross-fire of interactions has been discovered. If the first eight cells are separated into couples, as in Fig. 7. 4a, devclopment is not fully mosaic, since no brain, adhesive organs or eye-spots appear in the larva from the anterior animal couple, nor any spinal cord from the anterior vegetative couple. The other experiments summarised in that Figure show that the brain, eyes and adhesive organs are induced to develop from the anterior animal cells by some influence proceeding from the anterior vegetative couple, but this influence is not effective on posterior animal cells. In the standard embryological terminology, we should say that only the anterior animal cells are competent to react to the stimulus from the anterior vegetative blastomeres. There is some evidence that the interaction is still more complex, in that there may be an influence from the posterior vegetative blastomeres tending to inhibit the development of the brain in the cells immediately above, even if these are an anterior couple. The formation of the spinal cord of the nervous system is also dependent on reactions between different regions, but in this case it is a derivative of the anterior vegetative blastomeres which does not develop when isolated from the anterior animal cells.
We therefore find that, far from the ascidian egg being a complete mosaic, it first undergoes a period in which a number of ooplasms become localised, and then enters one in which, although several of the regions are already endowed with a capacity for independent differentiation, some others are still labile, and develop normally only if certain reactions take place. This lability affects particularly the nervous system, which has two rather distinct parts, the brain and spinal cord, while the eyespots and adhesive organs also become fixed in their developmental fate at the same time. We shall see that in vertebrate embryos the development of the nervous system, and of many other organs which depend on it, is ones mesoderm, including muscle, and endoderm; this corresponds with their prospective fate. But the anterior animal ones give only ectoderm, the result of a process of induction, and we can see the first signs of this in the ascidians. But in the ascidians the primary inductions occur during the early cleavage divisions, while in the higher vertebrates they happen much later.
File:Waddington1956 fig7.4.jpg
Figure 7.4. Isolation and grafting of blastomeres in ascidians. (a) Separation of the 8-cell stage of an ascidian into four pairs of cells. The two posterior animal cells give ectoderm, and the two posterior vegetative no brain or palps, and the vegetative anteriors endoderm and notochord but no neural tube. (b) If the anterior vegetative cells are removed, the rest does not develop the brain and palps (although the anterior animal cells from which they should form are still present), nor neural tube. (c) Ifthe anterior animal cells are removed, and the brain and palps are again absent. (4) The anterior animal couple are removed and replaced by a posterior animal couple (shaded). Still the brain and palps are absent. (After Reverberi 1948.)
Suggested Reading
The papers of Conklin 1905, 1932, are classics of descriptive embryology. In addition Rose 1939, Reverberi 1948, Dalcq 1938, pp. 103-27.
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