Waddington1956 9
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Waddington CH. Principles of Embryology (1956) The MacMillan Co., New York
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Chapter IX The Vertebrates: The Amphibia and Birds
The group of vertebrates contains a variety of types which are sufliciently diverse to exhibit most of the important principles of embryology, but are not so bewilderingly various as to obscure the fundamental plan of which they are all modifications. They are therefore peculiarly suited for comparative study. Moreover, the rather large size of many vertebrate eggs has made them favourite objects for experimental analysis, and our understanding of the epigenetics of the group is at least as great as that of any of the invertebrate phyla. This is particularly true of the Amphibia, and only slightly less so of the birds. These two groups have for long been classical teaching material, since frogs’ and chickens’ eggs are some of the easiest to obtain for students’ use. The discussion of vertebrate development in this book will be largely based on these same two objects, although as an amphibian type, the newt’s egg will be referred to perhaps more often than that of the frog, since, although they are basically similar in the characters of interest in an elementary account, the former shows these features in a somewhat clearer way; moreover, for technical reasons connected with case of manipulation, it has proved more favourable than the frog egg for experimentation. It is only after the early development of these two types has been described and discussed that we shall turn to consider, more shortly, the embryology of the other vertebrate phyla.
Detailed descriptions of the development of Amphibia are to be found in many general embryological textbooks, particularly Dalcq and Gerard (1935). For the chick there are several special monographs. Patten (1950) and Huettner (1949) are good descriptive texts, the figures in the latter being particularly clear; Hamilton’s (1952) revision of Lillie is the most complete descriptive monograph, but tends to neglect non-American work; Waddington (19524) deals mainly with the carly stages of development, and with experimental studies. Details of most microsurgical techniques are given by Hamburger (1942) and Rugh (1948), but neither of them deals with organiser grafts in birds.
1. From the unfertilised egg to the formation of the blastula
(a) The Amphibia
The fully grown frog’s or newt’s egg is a fairly large spherical cell, some 2 or 3 mm. in diameter. It is usually seen after being extruded into the water; normally the eggs are fertilised either as they leave the female’s body (in frogs) or by sperm which the female has taken up into her cloaca (in newts). The egg within the ovary and oviduct is surrounded by a viscid layer of jelly, which swells on contact with water into a thick protective covering; in newts each egg is enclosed within a separate capsule, but in frogs a whole clutch of eggs coheres into a single mass within which the individual eggs are scattered.
Amphibian eggs contain moderate quantities of yolk; much more than non-yolky types such as echinoderms or ascidians, much less than the” extremely yolky birds’ eggs. The yolk is present in the form of small platelets or ovoid granules, and, although scattered throughout the whole egg, is more concentrated at the vegetative end where the granules are also larger in size. Near the opposite, animal, pole is a large germinal vesicle filled with clear sap. The heavy load of yolk granules makes it rather difficult to distinguish different regions of cytoplasm, such as those so well shown in the ascidians, and it is only recently that the earliest phases of development are becoming clear (Reviews: Pasteels 1951; Dalcq 1950b). There is, for instance, always a peripheral zone or cortex which contains little yolk; the animal half of it usually carries a considerable number of pigment granules, which make up a dark animal cap which is very clear in the frog; the outermost layer of all is a relatively impervious, very extensible membrane, the ‘coat’ (Holtfreter 19434). In the interior of the egg, it is usually possible after the germinal vesicle has broken down to distinguish a clearer animal plasm, in which the yolk granules are smaller and more scattered; this appearance may be duc to the admixture of the nuclear sap (Fig. 9.1). In some forms (the frog and axolotl) there is also a darker ‘marginal plasm’ which lies in a circle fairly close under the surface just above the equator, and in the frog there is another, central, region with small yolk platelets (Pastecls 1951).
The pattern of cytoplasmic regions in the unfertilised egg is thus radially symmetrical and shows no obvious sign of the future dorso-ventral plane of symmetry. An indication of this plane does, however, appear fairly soon after fertilisation in many amphibian eggs. The first apparent result of fertilisation is a slight lifting-off of the inner or vitelline membrane from the egg, which thus becomes free to revolve within its jelly capsule and lie in its natural position with the heavy yolk-laden vegetative pole downwards; the swing round into this position takes only a few minutes. Soon afterwards, in the frog and some other species, a “grey crescent’ appears on one side, lying between the dark animal hemisphere and the pale yolky vegetative end. This is destined to play a fundamental part in later development. Its fate cannot be followed without special methods, since as the egg cleaves up into many cells, the even colour of the animal pole becomes broken up by the cell boundaries so that it merges into the paler tint of the crescent, which ceases to be recognisable. But we can apply the technique of vital stained marks (p. 158). A small spot of colour placed in the centre of the grey crescent is found eventually to lie in the dorsal midline of the animal, and, in fact, somewhere in its notochord. The grey crescent therefore marks the dorsal side, and, in its position, corresponds to the grey chorda-neural crescent which appears, at a somewhat later stage, in the ascidian egg.
FIGURE 9.1
Ooplasmic regions of the unfertilised ege of Xenopus. The main regions are: the animal region (Z.AN.); the marginal zone (Z.M.); the central region (N.C.) and the vegetative region (Z.Veg.). The upper drawing shows the location of these in a transverse section, while the lower drawings illustrate the nature of the cytoplasms. (From Pasteels 1951.)
The appearance of the grey crescent and the marking of the dorsal side is the first great step in embryonic development in the Amphibia. Naturally it is important to know its causal antecedents and its causal consequences; what brings it about, and what effect does it have? There is general agreement about the latter. After the grey crescent has appeared, every developmental performance of the egg is related to it. If, for instance, the egg is cut in half (which can be done even before the first cleavage by putting the egg into a loop of fine hair which is slowly pulled tight (Fig. 9.2), then only those halves which contain some grey crescent material will develop any of the main embryonic tissues, such as neural system, notochord, somites, kidney, etc.; ventral portions of the egg which have no crescent material, usually form only skin and disorganised mesoderm and endoderm ‘not recognisable as any definite tissue, though Dollander (1950) has recently shown that in some cases a certain degree of regulation occurs and the ventral parts also produce a little neural tissue and axial mesoderm. The grey crescent, in fact, is the precursor of the ‘organisation centre’ of the gastrula, which, as we shall see, (p. 175) is the agent which causes the formation of the rest of the embryo.
The antecedents of the grey crescent are less well understood (Ancel and Vintemberger 1948, Pasteels 1951). Its position is certainly not completely fixed before fertilisation, since it is possible by suitable treatment to make it appear in any desired meridian of the unfertilised egg. If, for instance, an egg is fertilised with sperm brought on a needle to a given place on the surface, the grey crescent usually appears at or near ‘the diametrically opposite side. Similarly, if the newly fertilised egg is held for some time so that one side of the yolky hemisphere is much higher than the other, the grey crescent usually appears on the higher side.
The experiments of the last paragraph demonstrate that the grey crescent is not fixed before fertilisation, or indeed much before it actually appears. But the same experiments also suggest that there is a pre-disposition of a certain plane to become the plane of symmetry. The experiments of tilting the egg, or of fertilising it in a definite place, do not always determine the position of the crescent; the cases in which they fail are those in which they do not overcome the existing predisposition. Again, in normal development there scems to be a tendency for the sperm to enter the egg in a predetermined plane, so that its influence on the position of the crescent usually reinforces a prior condition. Finally, when newly fertilised eggs are constricted into two halves with a hair loop, we find that certain halves, although they contain the nucleus, develop into featureless lumps exactly similar to those, derived from a later stage,;which contain no grey crescent material; and this indicates that, even immediately after fertilisation, the crescent material is to some extent localised so that egg fragments may contain none, or too little, of it.
FIGURE 9.2
A newt’s egg, in its jelly capsule, is constricted into a dumb-bell shape soon after fertilisation. A, cleavage occurs first in the portion containing the nucleus; B, after some time a nucleus passes through the stalk, after which cleavage begins in the other (left) portion also; C, both parts may give rise to a complete embryo. (From Schleip 1929, after Spemann.)
In recent times, Pasteels is the author who has studied in most detail the relation between the grey crescent and the various plasmatic regions of the egg (see his review, 1951). The internal contents of an amphibian egg are fairly fluid and if the egg is turned upside down and held with the vegetative pole uppermost, the heavy, yolky material from that end streams down and comes to lie against the cortex near the animal pole region. The grey crescent is not distinct enough in appearance always to be recognisable with certainty in such eggs, but as we have seen, in later development it gives rise to the blastopore and that structure, at least, is unmistakable. It is found that blastopores always appear at the edge of regions in which masses of yolky cytoplasm are in contact with the cortex (Fig. 9.4). At these blastopore regions invagination takes place and an embryo will eventually develop. Its cephalo-caudal polarity is determined by the gradient in yolk content, the future head end always originally lying nearest to the most concentrated mass of yolky cytoplasm. It is clear from these experiments that the position of the grey crescent is determined by the mutual relations of the yolky cytoplasm and the cortex.
In eggs which have been held upside down and in which drastic alterations of the internal structure have occurred, it is difficult to be certain of the nature of the interaction which takes place between the yolky cytoplasm and the cortex. In the normal egg Ancel and Vintemberger have shown that the formation of the grey crescent involves a movement of the cortical layer of that region towards the animal pole (Fig. 9.3B). This seems to carry up with it some of the underlying yolky cytoplasm which thus becomes thoroughly mingled with the marginal cytoplasm with its larger content of basophylic granules and mitochondria. It seems probable that it is this mingling of yolky and marginal cytoplasm which is the essential feature of the grey crescent. If shortly after fertilisation and before the appearance of the normal grey crescent an egg is tilted slightly so that the animal-vegetative axis makes an angle with the vertical, the yolky cytoplasm slides down into the lowest position and in doing so leaves behind it a sub-cortical layer which has much the same appearance as that of a normal grey crescent: and as we have seen it eventually develops into a blastopore. This experiment was one of the earliest to be carried out in amphibian experimental embryology. It was originally performed by Born in the eighties of the last century. It has recently been studied in detail by Pasteels (1951). Pasteels also shows what happens when the experiment is carried out slightly later, after the appearance of the grey crescent. In eggs rotated at this time the blastopore will, again, eventually appear somewhere at the edge of the main mass of yolky cytoplasm, and presumably it was preceded by the formation of something corresponding to a grey crescent, although it is not always possible to recognise this clearly. The position of the blastopore and of the putative grey crescent is now, however, not always determined by the direction in which the yolk slid down to the bottom of the egg. It is influenced rather by the position of the original grey crescent which had formed before the egg was tilted. The blastopore appears at that edge of the yolky mass which is nearest to this position (Fig. 9.34).
FIGURE 9.3
A. The effects of partial rotation of the uncleaved axolotl egg round an axis perpendicular to the dorso-ventral plane. The small arrow points to the grey crescent and the dots indicate the heavy vegetative odplasm. Note that the blastopore always appears at the margin of this, in whatever position is nearest to the original grey crescent. (From Pasteels 1951.) B. The movement of marks on the cortex towards the animal pole (P.a.) during the formation of the grey crescent. (From Pasteels 1951, after Ancel and Vintenberger 1948.)
Pasteels (see also Dalcq 1950b) concludes that the properties of the grey crescent and thus eventually of the blastopore are brought about by the interaction of two factors. The first is a gradient in cytoplasmic constitution which normally runs from the animal pole (rich in cytoplasm and cytoplasmic granules, and poor in yolk) to the vegetative pole (large yolk platelets and little cytoplasm). This gradient determines the cephalocaudal polarity of the embryo which will develop. It interacts with a cortical field which has a point of highest activity in the future grey crescent region and falls off from that in all directions (Fig. 2.7, p. 42). It has been necessary to dwell at some length on these early events since the determination of the plane of bilateral symmetry is, in many ways, of considerably greater importance than anything which happens in the much more striking phenomena of cleavage. Amphibian eggs, having a moderate charge of yolk, undergo cleavages which are definitely, but not exaggeratedly, unequal. The first cleavage plane is vertical, and usually, though not always, coincides with the plane of bilateral symmetry running through the middle of the grey crescent—a coincidence brought about by the fact that the sperm has an influence, again considerable but not quite always effective, on the plane of the first cleavage, just as it has on the plane of the grey crescent. The second cleavage is also vertical, and perpendicular to the first. In many species, the first two dorsal cells are smaller than the two ventral ones, which is an indication that the cleavages are based on a bilateral-symmetrical pattern, which seems to underlie all vertebrate cleavages, although it is often difficult to distinguish.
FIGURE 9.4
The relation between the main yolk-mass (dotted) and the anterior-poster ior axis of embryos developed following rotation of the frog’s egg. The arrows point to the cephalic region of the mesoderm; a is the normal situa tion; b, c and d show inverted eggs in which there is little, considerable or complete redistribution of the yolk to lower pole. Note that the cephalic region of the mesoderm (i.e. the blastopore) always forms near the yolk~ mass. (From Pasteels 1951.)
The third cleavage is horizontal, and the furrow lies above the equator, so that the animal cells are smaller than the vegetative. This is the first indication of the effect of the yolk gradient, whose influence is predominant throughout the remainder of the cleavages. These soon become irregular, and proceed faster in the animal than the vegetative region, so that the difference in volume of the cells becomes progressively more marked. At an early stage—about the fourth or fifth cleavage—a space appears in the centre of the mass of cells, the so-called cleavage cavity or blastocoel. This, of course, lies above the equatorial plane, and, as the unequal cleavages proceed, it not only increases in size, but shifts further and further towards the animal pole.
Cell division continues throughout the whole of embryonic development, but the ‘period of cleavage’ is considered to end when something else begins to happen. The first definite event which occurs to terminate it is the appearance of the blastopore and the beginning of gastrulation. By this time the egg, which at this stage is called the blastula, has become a hollow ball, with a thin roof of animal cells covering a large cleavage cavity or blastocoel, beneath which lies a floor of large yolky blastomeres.
The most important processes which have been going on under cover of the cleavages have been two. Firstly, the divisions have cut up the egg into cells of a size more attuned to that of the nuclei; and there has been a considerable increase in the total amount of nuclear material, and perhaps a synthesis of DNA, to assist in bringing nucleus and cytoplasm back to their normal relations (but see p. 58). Secondly, there has been a considerable movement of material; if vitally stained marks are made on the vegetative pole of the egg, the dye is gradually carried right into the body of the egg, and eventually reaches the floor of the blastocoel.
The significance of this movement is not yet fully understood (Nicholas 1945). It does not, however, affect the very thin coat which forms the actual surface of the egg. This material, which has special properties of elasticity and toughness, remains on the exterior surface forming the boundary between the egg and the external medium, and seems to play an important part in the biophysics of the morphological changes which lead to the formation of the embryo (p. 439).
The first sign of gastrulation is the appearance of a shallow groove, the blastopore. This lies somewhat below the equator and within the area of light-coloured yolky cells. As was said above, vital staining shows that it appears in the region of the egg derived from the grey crescent, which by this time is no longer recognisable. Before describing the later events, we shall follow the development of the bird embryo up to the corresponding stage, so as to be in a position to compare the gastrulation of the two forms.
(b) The birds
A discussion of the development of the bird’s egg up to the blastula stage will take much less space than was required for the amphibian. This is not because the events are less complex, but because our knowledge of them is less complete. The early stages of avian development remain difficult to explore, partly because the enormous stores of yolk obscure any cytoplasmic differentiation there may be, and partly because the egg at this stage is out of easy reach within the body of its mother, who does not lay it until the cleavage period is finished and the gastrulation begun.
The true ovum of a bird such as the chick does not make up the whole of what we usually call the egg, but only the so-called yolk. This is covered with a well-defined and tough membrane—the vitelline membrane—outside which lies the albumen or ‘white’ which is again enclosed in a membrane, the whole being finally covered by the shell. All these parts, from the vitelline membrane outwards, are non-living additions to the egg-cell, serving as sources of nourishment or means of protection; they are laid down around the ovum after it has left the ovary, been fertilised, and is on its way down the oviduct. The ‘yolk’ or true egg-cell is not adequately described by its popular name, since although it obviously contains a very large quantity of yolk, this is by no means the whole or the most important part of it. At one point (which lies in the plane of the smallest section of the ovoid egg) there is a small area of clear cytoplasm containing the nucleus. It is from this that the whole embryo is derived.
The great stores of yolk affect development from the very beginning. Whereas in most eggs, the penetration of one sperm suffices to prevent the entry of a second, in highly yolky eggs such as birds’ this mechanism breaks down, and a number of sperm penetrate. Only one of these completes fertilisation by fusing with the egg nucleus; the remainder form subsidiary nuclei which probably play a part in digesting the yolk in the very early stages of development. In the next events, those of cleavage, the influence of the yolk is even greater. The cleavage furrows start in the clear cytoplasmic region, and never succeed in forcing their way down into the inert mass of yolk. The first two cleavage planes are vertical, but the third, while also starting as a vertical furrow in the flat layer of cytoplasm, soon curves round so as to run horizontally parallel to the surface, and thus cuts underneath the cytoplasm and separates it from the yolk. The cleavage pattern, even in these very early stages, is irregular, and we have little exact knowledge of how the cleavage planes are related to later development.
The cleavages convert the cytoplasmic region into a small compact plate several cells thick. The cells contain fairly large quantities of yolk granules, and at the edges the plate merges into the uncleaved yolk through a zone of increasingly large and more yolky cells. Beneath the mass, however, the yolk begins to liquify, and in sections this region appears as an empty space, the ‘subgerminal cavity’. There is considerable debate as to exactly what happens next; and on this turns the question of whether the subgerminal cavity is considered to be equivalent to the blastocoel cavity of the amphibian egg or as a mere local modification of the yolk. The thin plate of cells, lying on the massive yolk, is very easily shrunken and distorted by normal histological methods of fixation and presents great difficulties to the experimentalist; it will not be until ways are found of overcoming these that we shall reach a fully satisfactory interpretation of events. At present, one of the main views (held by Pastcels 1936-7, Peter 1938 and others) is that the subgerminal cavity is not equivalent to the blastocoel, but that the latter soon begins to appear in the form of irregular horizontal splits within the mass of cells; these cavities gradually expand and run together until they form a thin space separating an upper from a lower layer. This space would then be the blastocoel, and the lower layer of cells would correspond with the large yolk-laden cells at the vegetative end of an amphibian blastula. In the bird, these authors hold, the lower layer merely stays where it is during gastrulation and forms the endoderm (Fig 4.1, p. 59).
Others believe that the endoderm, instead of merely splitting off from the upper layer, is derived from it by a more active process which can be regarded as a modified invagination; and they therefore consider the subgerminal cavity, into which the endoderm is pushed, as a true blastocoel.
Different adherents of this view, however, have very different ideas as to the nature of the active process by which the endoderm originates. Perhaps the simplest of these is that of Patterson (1909), who supposed that the posterior edge of the cellular plate becomes folded under and grows forward below the remaining part. This has become one of the commonest accounts given in textbooks, perhaps because of its apparent simplicity; but, unfortunately, the evidence for it is negligible, and all authors who have examined the matter for the last forty years (except Lutz 1953, 1954) have denied it. Another, more plausible, view is that isolated cells are pushed out from the cellular plate and gradually build up a lower layer. Jacobson (1938) believes that endoderm formation begins in this way, but that the process goes on fastest in the posterior region (though not quite at the posterior margin) and eventually attains such an impetus there that the whole plate is bent down into a groove and migrates en masse into the endoderm. He describes the formation of a centre of invagination which would really merit the name of a blastopore and closely resembles the structures which, we shall see, are undoubtedly formed at a much later stage when the invagination of mesoderm occurs. Most later authors have been unable to confirm Jacobson’s account in full. A final view (Hunt 1937) which must be mentioned is that, however the lower layer of cells is produced at this stage, it eventually does not form the endodermal organs of the embryo, such as the gut, but is at a later stage pushed out to the sides by cells which come out of the upper layer (from the primitive streak, which forms there, see later).
Although it is not possible to decide finally between these possibilities at the present time, the safest view is probably that the greater part of the endoderm is formed in the first way mentioned, by a splitting or delamination of the original cell mass. On this interpretation the blastocoel is not represented by the subgerminal cavity, but by the cleft which separates the upper from the lower layer.
During the period when the lower layer is forming, the mass of cells is also becoming thinner and spreading more widely over the surface of the yolk. From this time on it is usually known as a blastoderm or blastodisc, and its upper and lower layers are frequently—and non-commitally, a useful point in the circumstances—referred to as the epiblast and hypoblast respectively. Moreover, a certain differentiation is appearing in plan. In the more central part, the cells are beginning to have used up their content of yolk granules, so as to become more transparent, while all round the periphery they remain heavily charged and opaque. As the subgerminal cavity becomes more definite below the central region, this differentiation into two concentric zones increases until there is a well marked central area pellucida surrounded by an area opaca. The embryo is formed entirely within the former. The opaque area is concerned mainly with digesting and liquefying the yolk, a process which is carried out chiefly by the underlying cells, which in this region do not separate cleanly from the upper layer, so that the whole zone remains a mass of rather spongy tissue not clearly divided into an epiblast and hypoblast till somewhat later.
By the time the egg is laid, in most birds (such as the chick), the blastoderm has already differentiated into a fairly well-defined area opaca and area pellucida, and, in the latter, the hypoblast and epiblast are well separated from one another, except perhaps in the anterior region, where the hypoblast may not yet have appeared. Very shortly after this, a thickening appears in the posterior region of the area pellucida. This is the beginning of the primitive streak, and the first sign of gastrulation.
2. Gastrulation: presumptive maps
At the beginning of gastrulation, the embryos of Amphibia and birds present completely different appearances. The former is a hollow sphere, with a thin roof and a thick floor surrounding a large blastocoel cavity; the centre of gastrulation is indicated by a blastopore, at this stage a small crescent-shaped groove lying within the area of pale yolk-laden cells of the blastocoel floor. The bird embryo is in the form of a blastoderm, a thin sheet of cells floating over a subgerminal cavity filled with liquified yolk; the blastocoel is represented only by a narrow cleft within the sheet, dividing it into an epiblast above and a hypoblast below; and the centre of gastrulation is indicated by the ‘primitive streak’, a short linear thickening in the posterior region of the area pellucida. The end-products of gastrulation in both forms are, however, similar in many essential respects. Both contain three layers of tissue, a mesoderm lying between an outer ectoderm and an inner endoderm. In both an embryonic axis is beginning to develop. An axial strand of ectoderm is thickening and folding up into a tube to become the rudiment of the central nervous system. Immediately beneath this, mesoderm is forming a notochof the.long narrow rod of tissue; while on either side of the notochord the ther of mesoderm is condensing into a row of separate more or less cvf vial blocks, the somites. And beneath the mesoderm again, the endoderde is also forming an axial tube, the rudiment of the gut. The movemeia: and foldings by which these two dissimilar starting-points can be,orought to these two similar end-products must necessarily differ in .ny ways; but there is one further point of resemblance which has not yet been pointed out, and which renders the two forms much more easily comparable and understandable. This lies in the maps of presumptive? fate; if we take the centres of gastrulation as our points of reference, these have a similarity which the crude comparison of the blastula and the blastoderm does not exhibit.
Amphibian eggs were the first to which the technique of vital marking was systematically applied to discover the map of presumptive area. The method consists in pressing against some region of the egg a small lump of agar impregnated with a dye which will colour the cells without doing them any great damage. The coloured patch can then be followed through its later development, and its fate ascertained (Fig. 9.5). This was done by the German embryologist W. Vogt in 1925, and his results, summarised in 1929, have remained substantially unchanged ever since, although many later authors h.O! 1 vised them in detail (Fig. 9.6).
FIGURE 9.5
The movement of vitally stained marks towards the blastopore, and thence into the mesoderm; e shows the location of the material in a transverse section of the neural plate stage. (From Vogt 1929.)
Vogt showed that “lay whole lower part of the blastula becomes endoderm and forms th"Cut and its annexes; the whole upper hemisphere becomes ectoderm, diffe: itiating into the nervous system and the epidermis with its derivatives Such as the ears, nasal placodes, etc. In between these two lies a broad belt w2 -h forms the mesoderm; on the dorsal side, immediately above the blastopore (and thus in the location of the old grey crescent), this develops into notochord; below this region, and further to the side, lie two areas which become the two rows of mesodermal somites, while the remainder of the belt forms the rest of the mesoderm (side-plate, tail-bud, etc.). Thus, if we look at the map from the blastopore outwards, we find first a zone of endoderm within which the young blastopore lies, then a ring of mesoderm, and finally the ectoderm.
- 1 The word used by the German authors who originally made such maps was ‘pra sumptive’, and many English authors use ‘presumptive’ for this: American writers, however, tend to use ‘prospective’.
The elucidation of the presumptive map in the birds was by no means so simple technically as in Amphibia (summarised: Waddington 1952a).
FIGURE 9.6
Map of the presumptive areas of a urodele gastrula. The blastopore BI lies in an area of endoderm (in which the gill slits of the pharynx are indicated). Immediately anterior to it is a wide region of presumptive notochord (dotted), and on either side of that somitic mesoderm (lined), which passes off into ventral mesoderm (unshaded). The animal half of the egg is taken up by presumptive neural plate (dashed), and epidermis (unshaded). On the left, seen from the side, on the right from the dorsal surface. (From Pasteels 1940.)
A beginning was made by Wetzel in 1929, who was able to stain small regions of the blastoderm in the opened egg; Griper, at about the same time, made speeded-up stereographic cinema films through a window in the shell, from which similar conclusions emerged; and in the next few years, Waddington was able to check many of their suggestions by experiments made on blastoderms removed from the shell and cultivated in tissue culture. Improvements of the methods of vital staining were made by Pasteels (1936-7); and Spratt (1946) has developed a technique of marking parts of the blastoderm with carbon particles, which allows of a more precise labelling, but can only be done on embryos cultivated in vitro.
The results of studies by vital staining in ovo and by carbon marking in vitro have yielded very different maps for the early gastrula, i.e. for the stage at which the streak is just forming. These are shown in Fig. 9.7. The main difference arises from the fact that Pasteels, in his work with vital stains, finds that during the growth of the streak a considerable movement takes place towards the anterior, while this was not apparent in Spratt’s studies. Pasteels’ student Malan (1953) has recently examined the matter again, and it has been rather convincingly shown that the absence of this movement in Spratt’s material is due to the abnormal conditions of the in vitro culture, to which the early stages are particularly susceptible. Thus although Spratt’s map has been accepted by most recent American authors (e.g. Hamilton 1952, Patten 1950, Rudnick 1948), Pasteels’ earlier one is probably nearer the truth.
FIGURE 9.7
Maps of presumptive areas in the chick. On the left, just before the streak appears, according to Pasteels, above, and according to Spratt, below (taken from Malan 1953 and Hamilton 1952 respectively). On the right two stages in the formation of the streak (from Waddington 1952a). Epidermis, white; neural tissue, vertical lines; notochord, dotted; axial mesoderm, close horizontal lines; lateral mesoderm spaced horizontal lines (the very widely spaced lines in Hamilton’s map are extra-embryonic mesoderm); already invaginated mesoderm, crosses.
In considering this early stage, we are faced, however, with another uncertainty. What is the presumptive fate of the very early primitive streak? Vital staining has shown that in its later stages the streak consists of presumptive mesoderm. But there are great technical difficulties in making critical experiments of this kind on the early stages, and it remains perfectly possible that there is some presumptive endoderm still remaining in the streak when it first forms; this would certainly be so if Jacobson’s account of endoderm formation (see p. 156) were accepted. There is little doubt that in reptiles (p. 234) the endoderm comes from somewhere in this general region of the embryo, and if in birds it originates from a definite part of the surface of the original cell-plate, this must be the place; but, of course, if it arises entirely by delamination from the lower part of mass of cells, there would be no definite location for it on a presumptive map of the surface.
We are on firmer ground when we turn to the other tissues. Vital marks have clearly shown that most, if not all, the primitive streak and the area on each side of it becomes mesoderm, while the area further away takes part in the formation of the ectoderm; the prospective neural ectoderm lies anterior to the mesoderm near the midline.
Comparing the maps of Amphibia and birds, one sees that their general pattern would be similar if one might suppose that the amphibian gastrula has been opened at some point within the area of skin ectoderm, and this hole enlarged until the original map was flattened out to a circle. We should then have an area of endoderm immediately round the blastopore, surrounded by a ring of mesoderm fringing which is an outer ring of ectoderm, with the neural ectoderm concentrated at one end, the anterior. This is exactly the picture we should find in the early primitive streak stage of birds if we suppose that any endoderm originates from the surface. A fuller discussion of the relations between the two groups is given on p. 243.
3. The gastrulation movements
(a) Amphibia
We have now described the state of affairs at the outset of gastrulation and given a sketch of the condition at the end of it. The process of gastrulation consists in the set of movements and foldings which convert the former into the latter. It is clear that, since the bird and amphibian gastrulae differ so considerably, while the early embryos possess roughly similar organs, the two processes must take different courses. These must now be summarised.
Gastrulation in the amphibian is the simpler of the two. It has been followed in great detail by the vital staining technique, but for our purposes it is only necessary to consider the general outlines of events. Before the advent of the staining method, the gastrulation process could only be inferred by comparison of a series of fixed and stained preparations of successive stages. It was natural to try to build up a picture in terms of foldings, delaminations and localised growth; and a series of technical terms, such as ‘epiboly’, ‘emboly’, ‘invagination’, ‘involution’, etc. were employed in this connection. The newer methods showed that the process is actually quite different from anything which had been envisaged. The most fundamental type of gastrulation movement is a flowing or streaming one, in which a piece of tissue is carried bodily into a new situation. With the recognition of this fact, the older terms were seen to be not very appropriate, and they have largely disappeared from the literature. An exception may be made for the word ‘invagination’, which originally meant a massive infolding of a sheet of tissue (such as was described in the infolding of the endoderm in echinoderms (p. 82)), but which is now often used to cover any process by which an originally outer layer is moved into an interior position.
The most obvious change on the surface of the egg itself during the process of gastrulation is the growth, rounding up and final closure of the blastopore. When it first appears, this is a small somewhat pigmented depression lying beneath the equator within the endoderm. Fairly soon it enlarges laterally to form a short groove. This continues to grow longer, and its two ends curve round to a crescent shape, which passes on to a horseshoe and then to a closed oval. Although the blastopore originally lay wholly within the whitish endoderm, by the stage at which it has closed up to an oval, the more pigmented tissue of the animal hemisphere is found to be lying at its edge, so that the outside of the egg is completely dark except for the light spot of yolky cells within the oval blastopore; this is known as the ‘yolk-plug’ and is a very characteristic and easily recognisable feature. The history of the blastopore is, however, by no means complete; it gradually contracts in area, drawing together above the yolk-plug which is finally covered over and hidden from sight. By the time gastrulation proper is ended, and the first signs of the embryonic axis are appearing, the blastopore has been reduced to a narrow slit which runs in the direction of the embryonic axis.
By following through the history of vital marks, we can see that much more has been happening than the appearance of the blastopore would lead one to expect. Marks made anywhere within the ring of prospective mesoderm are seen to move down towards the edge of the blastopore, to flow over it, and, as can be shown by later dissection, to move away from it again underneath the surface. The lips of the blastopore are, in fact, not fixed positions occupied by a definite tissue, but are mere structural appearances showing where the flow of tissue along the surface turns downwards towards the interior of the egg. This explains how the lip, which was originally made up of the pale endoderm, later becomes lined with the darker material of the animal hemisphere; the pale material has already moved away inside, and the dark material has streamed down to replace it.
The streaming movement towards the blastopore begins when the blastopore is quite small; it always goes on fastest in the region in which the blastopore first appears, and the movement here involves a great stretching and elongation of tissue in the direction of the meridian joining the blastopore to the animal pole. This meridian will become the middorsal line of the embryonic axis when this begins to form; and this part of the blastopore is, therefore, known as its dorsal lip; the ventral lip is the last-formed portion which eventually appears on the diametrically opposed side of the yolk-plug. The material which flows in round the dorsal lip elongates considerably while doing so, and becomes narrower from side to side; this means that material invaginating further laterally has to move in towards the midline. The invagination streams, therefore, converge from the sides towards the middle, as shown in Fig. 9.8. Moreover, as the ring of prospective mesoderm moves into the interior of the egg, its place at the surface has to be taken by the prospective ectoderm. We shall see later that the prospective neural ectoderm also elongates along the midline, narrowing as it does so; thus, the whole of the dorsal convergence has to be compensated for by a lateral expansion of the prospective skin. In its crudest outline, therefore, the gastrulation movements of the animal hemisphere of the egg can be summarised as, firstly, a great elongation and narrowing along the dorsal meridian (the elongation being so much that the material flows round the blastopore and finishes up as a double layer), and, secondly, to compensate for this, a lateral expansion, in a plane at right angles to the dorsal one, of the prospective skin at the opposite side of the egg; with, of course, one process changing smoothly into the other as one goes from the dorsal midline towards the sides.
FIGURE 9.8 The gastrulation movements in a urodele, seen from the side.
The process we have described so far would give rise to only two layers, an outer which is the ectoderm and an inner which is the mesoderm. Within this again lies the endoderm, and we have as yet said little as to how it gets there. As a matter of fact, it is the endoderm which starts the whole movement, since the early blastopore lies wholely within it. At this stage, the invagination movement of the endoderm consists in the withdrawal inwards of the main bodies of some of the large endoderm cells, a process which can only be scen in sections (Fig. 20.13, p. 444). A little later the withdrawing endoderm is followed by the first mesoderm flowing round the dorsal lip. As this movement of the mesoderm spreads to more lateral regions, making the lateral lips of the blastopore, the edge of the mesoderm separates from the endoderm to form a free margin such as is pictured in Fig. 9.10; only in the mid-dorsal line is the connection permanently retained at the anterior of the archenteron, and here it has never been easy to decide where to draw the boundary between mesoderm and endoderm. The separation only works round slowly to the ventral side, with the gradual spreading of the blastopore lips. Meanwhile, the region of endoderm which has become free of the mesoderm behaves as though it were sucked in to the interior, folding inwards and at the same time elongating along the dorsal meridian to keep pace with the mesoderm to which its anterior end is still attached. Along the dorsal surface of the endoderm a groove appears, at first shallow, but gradually becoming deeper. This is the primitive gut, or archenteron. As is clear from a section (Fig. 9.9) its walls and floor are made of endoderm, but its roof is at first mesoderm—in fact, the mid-dorsal mesoderm which will later differentiate into the notochord.
Gastrulation is often said to be completed by the time the blastopore is reduced to a small slit. Although this is not actually the case, as can be seen from Fig. 9.10 (see also p. 263), it will be as well to pause in our account of it to notice some of the more obvious changes which begin to occur at this time. When the yolk-plug finally disappears from view, the egg is still spherical in shape, evenly coloured all over with the darker tint characteristic of the ectoderm, and diversified only by the blastopore slit, which is elongated in the meridian of the dorsal axis. Fairly soon after this, the first signs can be seen of the differentiation of the ectoderm into the neural system and the skin. The neural area lies immediately in front of the blastopore, and first appears (for instance in the newt, in which it is well exhibited at this stage) as a pear-shaped or dumbbell-shaped area of somewhat darker colour. The edges of this area soon become elevated as ridges, while between them the surface is somewhat flattened to make a wide shallow depression. The ridges which mark the boundary of the area are knownas the neural folds, while the depressed area between them is the neural plate. Sections show that the ncural plate is thicker than the remainder of the ectoderm (Fig. 20.21, p. 452). As time goes on this thickening increases, the plate simultaneously becoming narrower and the folds higher, until the whole neural area becomes more appropriately referred to as the neural groove rather than the neural plate. Eventually the neural folds approach so closely that their upper margins touch and fuse with one another; neural material joins on to neural, and skin to skin, so that the neural groove becomes converted into a tube lying beneath an unbroken covering of epidermis. Clearly the tendency of material in this region of the egg to converge towards the mid-dorsal line, which we noticed during the gastrulation movements, has continued even after the stretching in length has become less marked. The same is true of the underlying mesoderm. The central strip which overlies the primitive gut and forms its roof condenses together into a single median strand, the notochord; while the tissue which lies slightly more laterally also accumulates towards the midline, forming two strips of thickened mesoderm, which soon become segmented transversely, to form two rows of more or less cubical blocks, the somites.
FIGURE 9.9
Semi-diagrammatic drawings of newt gastrulae sectioned through the dorso-ventral plane. The ridge on the wall of the archenteron in c, d and e shows where the endoderm and mesoderm are separating from one another. (From Spemann 1938.)
All these changes begin at or near the anterior end, and progress steadily posteriorly; though it should be noticed that the neural groove, being originally wider at its anterior end, does not succeed in closing over so early in its widest parts as it does somewhat further back. At the most posterior end of the embryonic axis, the remains of the blastopore persist as a narrow slit; and here, as was hinted above, conditions are still much the same as they were in the more widely open blastopore of the yolk-plug stage, and gastrulation movements still proceed, although on a smaller scale. The notochord, at the dorsal apex of the blastopore, is the first to follow the endoderm completely below the surface, but the invagination of mesoderm round the lateral lips persists for some time longer. Vital staining demonstrates, in fact, that by the first appearance of the neural plate only the first dozen or so somites have been invaginated, and the mesoderm of the greater part of the trunk, and the whole of the tail, is formed from the small area which still remains on the surface (p. 264). The formation of these organs involves a great increase in length, and the tendency to axial elongation in the midline takes on a new lease of life in this slit-blastopore region. The focus of this elongation is, according to Pasteels, not quite at the blastopore itself, but slightly anterior to it, at the most posterior limit of the invaginated notochord.
FIGURE 9.10
Sagittal sections at three stages (a, b, c,) of gastrulation in a urodele, to show the expansion of the archenteron and the obliteration of the blastocoel; the extent of the lateral mesoderm is indicated by shading. Below, d is a dorsal view on to the blastopore (presumptive notochord closely dotted, presumptive neural system dashed); e¢ is a yolk plug stage; f, the first appearance of the neural plate; g, the neural fold stage. Note that in f considerable mesoderm, and even some notochord, is still on the surface, while even in g there is a little mesoderm between the neural folds near the remnant of the blastopore. (From Pasteels 1940, after Vogt and Nakamura.)
(b) Birds
Although the gastrulation movements in birds are more complex than in Amphibia, they are perhaps easier to visualise and to describe, since they occur not in a sphere, but in a flat circular disc. The main complication which is introduced is a double streaming movement along the midline, forwards at an early stage, and backwards later on. There is moreover another important difference from the Amphibia in connection with the type of growth which is proceeding. In the latter group the total mass of the egg is more or less constant during gastrulation; ‘growth’ consists in the divison of cells into smaller units, and the conversion into living substance of yolk, already contained in the cells. In the birds, on the other hand, the greater part of the yolk lies outside the tissues, and during gastrulation this is being digested and assimilated, so that growth involves an actual increase in the cellular mass.
We have seen (p. 155) that at the beginning of gastrulation in the chick a lower layer of cells is already present, formed either by delamination or by migration from a posterior region of the blastoderm. This remains mote or less in situ as the endoderm. It undergoes a general spreading out to cover the whole underside of the blastoderm, which may involve some posterior-to-anterior streaming, but if this occurs at all it is difficult to investigate by vital staining methods, and little is known about it.
The upper layer of the blastoderm consists of the presumptive mesoderm and ectoderm. (For which reason it is sometimes referred to as the mesectoderm, but since this word has been used in other senses, it is better to call it the epiblast.) The formation of mesoderm begins at an early stage. The site of its formation is the primitive streak, the linear thickened area whose appearance in the posterior part of the blastoderm we have taken as the signal for the beginning of gastrulation. At the primitive streak there is no open channel leading from the outside towards the interior, such as one finds at the amphibian blastopore. Nevertheless, vital markings show that tissue which originally lay on the outer surface of the epiblast streams from both sides towards the streak, turns downwards when it arrives there, and moves away again in a lower layer which lies beneath the surface but above the endoderm, and which is therefore the mesoderm (Fig. 9.11). The first mesoderm to pass through the streak migrates, not only laterally, but also towards the anterior. It does not form part of the mesoderm of the embryonic axis, but finally lies well out to the side. This is in strong contrast to what happens in Amphibia, where the first invaginated mesoderm remains in the dorsal axis. But the difference is merely one of the place at which the invagination of mesoderm begins; in the Amphibia this is the dorsal lip, while in the birds the first part of the primitive streak is in a region corresponding more nearly to the ventral or lateral lips.
FIGURE 9.11
Semi-diagrammatic section through the primitive streak of the chick, to show the invagination of mesoderm.
Very soon after, or perhaps even simultaneously with, the beginning of this mesoderm-invagination, another movement affects the region of the primitive streak. This is a streaming forwards along the streak towards its anterior tip. As this movement gets under way, the streak lengthens, and pushes out across the area pellucida. Whereas when it first appears it occupies only a fifth, or less, of the diameter, it eventually comes to extend more than halfway across the still circular area. While the forward movement is still continuing in the anterior region, the posterior part of the streak starts to push out backwards. When this double movement is at its height, the streak is elongating so fast that it draws out the area pellucida from its original circular shape into an oval or pear-shaped form (Fig. 9.12). The movement bends the regions of the prospective map into long arcs lying on each side of the streak, and since the invagination is continuing all the time, by the close of the forward streaming nearly all the lateral mesoderm has disappeared in the anterior region, leaving the streak bordered by prospective somite material, with a small arc of prospective chorda at the most anterior end (Fig. 9.7, p. 160). The anterior end of the streak becomes somewhat more markedly thickened than the remainder, and a slight depression is visible in the centre of it. The structure has been given the name of Hensen’s node, and a rather exaggerated importance was attached to it in the earlier literature, since some authors held that it alone was the analogue of the amphibian blastopore; actually, we have seen that mesoderm is invaginated throughout the length of the whole streak,
Hensen’s node does, however, mark the site of a somewhat special type of invagination. In most of the streak during the phase of forward streaming, the direction of movement of the mesoderm has been in towards the streak from both sides, and out again in the lower layer towards the sides and somewhat forwards, as indicated in Fig. 9.12. The formation of Hensen’s node seems to indicate the beginning of an invagination directed wholly along the midline; tissue, presumptive notochord to be explicit, moves from slightly anterior to the node backwards into it and after sinking to the lower level, passes out again directly forwards, so that a tongue of notochord extends anteriorly to the tip of the streak. This is known as the head process.
FIGURE 9.12
The tissue movements in the chick blastoderm: a, during the elongation of the streak; b, in the fully grown streak; c, during the regression of the streak. Solid arrows show the movements on the upper surface, dotted ones those at a lower level (i.e. in the mesoderm). (From Pasteels 1940.)
The formation of the head process brings to an end the forward movement along the embryonic axis, and from then onwards all the streaming movements are directed towards the posterior end. Although, as we saw, the backward movement started at an earlier stage in the posterior region, it soon acquires relatively greater speed in the neighbourhood of the node, which therefore travels back down the streak, catching up, as it were, the regions posterior to it. The node marks the most anterior point where invagination is still proceeding, and as it moves backward along the streak, the area in front of it is occupied by neural ectoderm in the upper layer and already-invaginated notochord and somites in the lower. Since the node is moving faster than the more posterior parts, the total length of the streak is continually being reduced; but it is a long time before the node finally overtakes the posterior tip of the streak and thus obliterates it altogether.
Before this happens the fundamental organs of the embryonic axis appear at the anterior end much as they did in the Amphibia. The plate of neural ectoderm rolls up into a groove and finally into a tube sunk below the epidermis; the underlying mesoderm separates itself into a median notochord flanked by thickened strips of somite material; and these become transversely segmented to form the paired cubical blocks of the somites themselves. The remnant of the streak which persists in the posterior all this time, where invagination is still proceeding, may be compared with the slit-like blastopore which we saw remains active while the neural groove is forming in the Amphibia; but in the birds the structure is not only relatively larger than in the frog, but continues in being to a stage in which the anterior part of the embryonic axis is much further advanced. The formation of a definite gut from the endoderm will be described later (p. 252).
4. General properties of gastrulation movements
We have confined ourselves so far to a straightforward description of the movements which carry the regions of the early gastrula into their final positions. These are the fundamental events by which the future animal acquires its organic form, and the biophysics of the process will be discussed in some detail later (Chapter XX). There are, however, some general points which may be mentioned here.
The forces producing gastrulation are not entirely functions of the egg as a whole, but are inherent in quite small parts of it. This emerges clearly from experiments in which parts of the gastrula are isolated. For instance, if in the stage with a fully formed primitive streak the chick blastoderm is cut transversely into two parts, the expected backward streaming along the axis takes place in both of them. This leads to the protrusion from the anterior part of a ‘tail’ containing the axial organs (neural tube, notochord and somites), while in the posterior half the medial material withdraws posteriorly, leaving a gap (Fig. 9.13). The same phenomenon can be seen even in smaller fragments when these are grafted into abnormal situations. Grafts taken from the region of presumptive mesoderm in the newt and placed in some other part of the gastrula, usually succeed in moving below the surface into the mesodermal layer, often forming a small blastopore of their own to do so. Moreover, the direction in which this invagination occurs is more or less definitely implicit in the fragment. Some of the most
FIGURE 9.13
Tissue movements in parts of gastrulae: a shows two vegetative half gastrulae of the newt placed together so that the blastopores point towards one another. The form which develops (6) is a‘duplicitas cruciata’; the two streams of mesoderm from the two blastopores have met head-on, and been forced to spread out to each side, so that each head region is derived half from one egg and half from the other. (From Schleip, after Spemann.) Figure ¢ shows the ‘tail’ developed from the anterior portion of a chick blastoderm transected just behind the node. (After Waddington 1932.)
striking examples of this have been described by Waddington (1941) in the anuran Discoglossus, in which the gastrulation movements are very rapid and perhaps for this reason seem to be rather definitely determined in the tissues; if a small part of the dorsal mesoderm of this form is removed and grafted back, reversed in direction, it starts pushing out a tongue of tissue which moves in the opposite direction to that of the main stream of mesoderm which surrounds it.
The inherent movement-tendencies of the parts of the gastrula are, however, at first by no means unalterable, as many experiments have shown. For instance if two gastrulae are cut in half transversely and the two halves containing the blastopores combined, the two forward-moving streams of axial mesoderm meet one another before they have completed their elongation. As Spemann showed (cf. 1938) they then combine and spread out to the two sides, so that finally double embryonic axes are formed in a cross-shape (a so-called Duplicitas cruciata). Again, if a fragment is taken from a region where the movement is not very intense (e.g. the lateral or ventral mesoderm in the newt, or the more posterior parts of the primitive streak in the chick) and is grafted near a region of active movement (such as the dorsal lip of the blastopore or the anterior primitive streak), the inherent tendencies of the graft often appear to be swamped by the more powerful tendencies of its new surroundings. And this does not seem to be merely a question of the graft being physically swept along in the tissue streams of its new location, but the phenomena suggest that the graft is as it were infected with the characteristics of its neighbourhood (Spemann and Geinitz 1927).
This infection of a graft with the dynamic tendencies of its surroundings has an important bearing on the classical problem of ‘the specificity of the germ-layers’. The older embryologists, relying entirely on descriptive methods of analysis, tended to reach the conclusion that ectoderm, mesoderm and endoderm were three fundamentally distinct types of tissue from a combination of which the embryo was built up. Soon after methods of experimental attack were discovered in Amphibia, however, Mangold (1925) showed that pieces of prospective ectoderm, if grafted into the mesoderm region in front of the blastopore, became invaginated with their surroundings, and thereafter behaved in every way as mesoderm, and also that ectoderm could be converted to endoderm in a similar way. In birds the demonstration is less complete, but Waddington and Taylor (1937) found that pieces of prospective ectoderm grafted into the primitive streak could become mesoderm, provided they became well enough assimilated to their surroundings for the coherent tissue to break down into single cells which migrate separately into the middle layer. These experiments show that there is no profound and permanent physiological difference between the three layers.
Suggested Reading
Vogt 1929 is a classical paper (in German, but the pictures should be studied). The early development of Amphibia is described in many texts (e.g. Nelsen 1953, Spemann 1928). Lehmann 1945, Fankhauser 1948, Dalcq 1950b, Pastcels, 1951 add important information on early stages. For the chick, Waddington 19524, Chapter 2, Hamilton 1953, Rudnick 1948.
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