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Chapter XI Embryo Formation in Other Groups of Vertebrates
Until fairly recently embryology has been in the main a comparative study. Its object has been conceived to be the derivation of a general scheme of development, of which the processes found in the different groups of animals could be regarded as modifications, In particular the diverse, but nevertheless obviously related, groups of vertebrates have provided fascinating material for study of the relations between different types of embryonic development. The wealth of available facts is indeed immense, and there are numerous textbooks devoted to it. Perhaps the best are Dalcq and Gérard’s revision of Brachet’s work and the recent book of Nelsen.
Comparative embryology of what may be called the classical kind was historically a derivative of, and theoretically should include, comparative anatomy. The comparisons instituted were between particular stages of the embryos of the various groups, each stage being considered as a static anatomical form.The results of such study are of great interest in relation to the general processes of evolution, though we consider them nowadays rather as raising problems than contributing materially to their solution. From the point of view on embryology which underlies the writing of this book, this aspect of biology remains rather peripheral, and since an adequate treatment of it requires the citation of an enormous number of detailed facts, we shall have to omit it and leave it in the hands of the comparative anatomists. The comparative method is still, however, a valuable tool of analysis. It can be applied not only to anatomical data but also to information of the kind with which this book has been mainly concerned. Such applications have only recently been attempted, but there are two types of comparative embryology which one must expect to increase greatly in importance in the near future. One is the comparative study of dynamic anatomical processes. Among the vertebrates there is a body of material which most urgently calls for treatment in this way, namely the active processes of gastrulation in the various groups which have been revealed by the use of vital staining or marking. Secondly, we now know enough about the causal sequences of epigenetic processes in the early development of the various groups to begin to institute comparisons between them, and to enquire what kind of alterations in these causal mechanisms have been produced by the processes of evolution. The importance of the comparative method has been acknowledged in the main chapters dealing with the vertebrates by treating in immediate juxtaposition the two best-known types, the amphibian and bird. In this chapter the field of comparison will be widened to include the other vertebrate groups. These will not be treated so fully, since our purpose here is not so much to obtain a detailed and thorough knowledge of the epigenctic processes in each group but rather to provide a sketch of the salient features for purposes of comparison.
Each group will first be discussed in turn, and in the last two sections of the chapter an attempt will be made to draw the parallels and point the differences between them.
1. Cyclostomes and other primitive fish
The eggs of a representative of the cyclostomes, the river lamprey (Lampetra or Petromyzon) have been studied in some detail in recent years. The egg is about 2 mm. in diameter. Its cleavage is total and very similar to that of the Amphibia. It gives rise to a blastula which is also very similar to the amphibian. At gastrulation a small knob begins to protrude in the spherical embryo and the blastopore appears just below it. The blastopore is small and remains so throughout the whole period of invagination and one sees nothing resembling the yolk plug of the frog.
Weissenberg (1934, 1936) has studied the gastrulation by means of vital stained marks, and has derived a map of presumptive areas (Fig. 11.1). It shows a considerable resemblance to that of the Amphibia except that the axial mesoderm is, from the beginning, more concentrated towards the dorsal plane. According to Weissenberg, indeed, the presumptive mesoderm does not extend right round the egg but on the ventral side the presumptive ectoderm comes into direct contact with the presumptive endoderm. This situation, which would constitute a considerable difference from that of the Amphibia, has been questioned by Pasteels (1940) who points out that in the early neural plate stage, when invagination is still proceeding around the blastopore, this structure is undoubtedly surrounded by a complete ring of mesoderm, some of which is being invaginated over the ventral lip at this time. Although Pasteels’ material was not sufficient to enable him to draw an alternative map of the presumptive areas in the early gastrula, he concludes that the mesoderm miust in fact extend right round the egg between the ectoderm and endoderm. He suggests that the main difference between the gastrulation of the lamprey and the frog is that, in the former, the invagination of mesoderm over the lateral and ventral lips is delayed so that it does not begin until the main mass of endodermal material has already passed into the interior. The two processes in fact differ only in this comparatively minor matter of timing. Some attention has also been paid to the causal embryology or epigenetics of these forms. It was shown as long ago as 1900 by Bataillon that if the first two blastomeres are separated cach may give a complete embryo. The experiment has been repeated more recently (Montalenti and Maccagno 1935; Bytinski-Salz 19372) and it has been shown that the situation is almost exactly the same as that revealed by constriction experiments in the Amphibia. Further, fragments from the dorsal lip of the blastopore can be grafted just as in newts (Bytinski-Salz 1937); Yamada 1938). When they come in contact with gastrula ectoderm they cause the induction of a secondary neural axis and, so far as experiment has gone, repeat in every way the behaviour we have seen in Urodele material. Unfortunately there has been little work by modern methods on other groups of primitive fish. Those types which have markedly telolecithal eggs and very unequal, but still total, cleavage would be particularly interesting as providing a transition to the higher forms, which are provided with very large masses of yolk and develop through the stage of a blastoderm. No vital stain experiments seem however to have been made on these transitional types. Ginsburg and Dettlaff (1944) have shortly reported a small number of experiments on the embryos of the sturgeon Acipenser. This has an egg, about 3 mm. across, which is rather amphibian-like in the cleavage and carly gastrulation stages. Grafts of the blastopore lip showed that it behaved as an organiser and could induce secondary neural folds in a host embryo. The donors from which the blastopore lip had been removed developed no nervous system or axial organs, and the authors suggest that the presumptive materials for these are confined to a smaller area than in amphibian eggs, although presumably located in a similar position.
Map of presumptive areas in the young gastrula of the lamprey (cyclostomes). Close dots, chorda; spaced dots, mesoderm (somitic mesoderm with horizontal lines); close vertical lines, neural plate; spaced vertical lines, epidermis; white endoderm. The position of the mesoderm on the ventral side is uncertain. (Modified from Weissenberg).
The gastrulation process has been studied in some detail in the dog-fish, Scyllium canicula, a selachian and therefore usually considered more primitive than the sturgeons (Vanderbrock 1936). Its eggs, however, are more like those of teleosts (higher bony fish). They are provided with a large quantity of yolk, and the cleavage is partial, giving rise eventually to a blastoderm. This gradually spreads over the whole surface of the yolk, and as it does so invagination takes place along one sector of the circumference, after which the neural folds appear in the same position. The blastoderm is a fairly thick structure, and much of the endoderm lies from the beginning in the depths of it beneath the surface. This is particularly true of the extra-embryonic endoderm; some of the material which will form the endoderm of the embryo proper is at first on the surface, lining the position where the blastopore will appear. It is the first material to be invaginated. The disposition of the other presumptive areas is shown in Fig. 11.3. One point worthy of note is that the presumptive mesoderm does not extend right round the blastoderm on to the ventral side, but is confined to the dorsal region in the neighbourhood of the blastopore.
The eggs of teleosts are rather large, and contain considerable quantities of yolk. Although there is extensive variation from species to species in the relative mass of yolk and of living cytoplasm, the cleavage is always partial and leads to the formation of a blastoderm. The structure of the egg at this time is not altogether simple. At the animal pole there is a relatively thick plate of well-defined cells forming the blastoderm proper. Beneath this is a cavity, and underneath that again and extending for some distance around the blastoderm, the yolk is admixed with a fair quantity of cytoplasm and contains scattered nuclei; this syncytium is known as the periblast. Covering the whole surface of the egg is a thin cytoplasmic membrane which shows the properties of low permeability, high contractability, and lack of adhesiveness on its external face, which are also seen in the external membrane of the amphibian egg. It may be referred to as the ‘coat’, using the word employed by Holtfreter for the similar structure in Amphibia. Where it lies over the yolk outside the limits of the blastoderm, it is also known as the ‘yolk gel membrane’. Its properties have been particularly studied by Devillers (1948) and Trinkaus (1949).
The blastoderm expands in area and after a time the sub-germinal cavity below it becomes well marked (Fig. 11.2). The maximum depth of the cavity which is roofed by the thinnest region in the blastoderm, EMBRYO FORMATION IN OTHER GROUPS OF VERTEBRATES 227
is not at the centre but is eccentrically based. This provides the first marked sign of bilaterality in the developing embryo. Vakaet (1953) has, however, found that the odcytes of the fish Lebistes show a bilateral structure when they are growing in the ovary. It may well be, therefore, that the dorsoventral plane in the blastoderm is foreshadowed at a much earlier stage.
A, B and C are lateral views showing three stages in the spreading of the blastoderm of the trout egg over the yolk. A’, B’, and C’ are views on to the blastoderm, which in the last two has been cut in order that it may be spread out flat. Bl, blastopore; e.a., embryonic area; g.r. germ ring.
Gastrulation (Reviews: Pastecls 1940, Oppenheimer 1947) takes place by the in-rolling of the margins of the blastoderm. This occurs all round the circumference, but goes on most rapidly at one point, which becomes the site of embryo formation. The endoderm is the first tissue to be invaginated. Its presumptive area lies as a thin band in the margin of the blastoderm, much of it already below the surface (Fig. 11.3). It is probably concentrated mainly in the posterior region from which the embryo will later arise, but its lateral and anterior extent is very inadequately known, and it may perhaps continue right round the whole margin of the blastoderm. Lying inside it, and the next material to be invaginated, is the mesoderm, which, according to Pasteels’ work on the trout, certainly continues round the whole margin. Inside this again, that is to say, towards the centre of the blastoderm, is the presumptive ectoderm. The shape of the presumptive neural plate described by Oppenheimer in Fundulus is quite different from that assigned to it by Pasteels in Salino, but the overall disposition of the areas is much the same in the two forms and their differences are perhaps such as we might expect from two not very closely related species.
Presumptive maps of the early gastrula (blastoderm) in the dogfish (Sela chia), and Fundulus and the trout (teleosts). Endoderm, circles; cephalic endoderm, small crosses; notochord, close dots; mesoderm, spaced dots (somites, horizontal lines); neural tissue, close reel lines; epidermis, spaced vertical lines. (After data of Vandebroek, Oppenheimer, Luther, and Pasteels.)
While the gastrulation is proceeding, the blastoderm is expanding rapidly, so as eventually to cover the whole yolk. Its margin is often thickened, forming a structure known as the germ ring. After the expanding blastoderm has passed the equator of the egg, the germ ring contracts and acquires a superficial resemblance to a yolk plug before it covers the yolk mass completely. The extra-embryonic part of the blastoderm later becomes vascularised, and forms the yolk-sac by which the embryo absorbs its nutrients from the yolk. While the blastoderm is still expanding and gastrulation continuing at its margin, the embryo begins to appear (Fig. 11.2). The connection between the embryo and the thickened germring caused many of the carlier embryologists to suppose that the two lateral halves of the embryo had originally been separated, one on each side, as though the embryo had been split from tail to head and the two halves pulled apart. These two half embryos were then supposed to come together and fuse in the midline by a process to which the name concrescence was given. It can, however, be seen from the map of presumptive areas that this is not the case. There is always complete continuity from one side to the other across the midline. The presumptive areas are wider from side to side than the definitive organs will eventually be, but the movement from one situation to the other involves only a lateral contraction and longitudinal stretching, and not a moving together and fusion of two originally separate rudiments (Fig. 11.4).
Gastrulation movements in the trout: 7 is an early stage, showing the whole blastodisc; 2 and 3 are stages in the formation of the embryonic axis. Move ments taking place in the surface layer shown in solid arrows, those occurring below the surface in dotted arrows. (From Pasteels 1940.)
The mechanism of the gastrulation movements, and in particular of the spread of the blastoderm over the yolk, has recently been extensively studied by Devillers (1951a) and Trinkaus (1951). They both agree, in contradiction to certain earlier authors, that the spread of the blastoderm is not solely due to the contraction of the yolk gel layer. It seems probable that one of the essential factors is the activity of the periblast, which seems to have a spontaneous capacity to spread over the surface of the yolk. The blastoderm also has an autonomous tendency to expand, but it only does so over regions which the periblast has already covered and thus provided with a suitable substratum for it.
Quite a number of studies have been made on the epigenetic mechanisms which bring about the development of the teleost (Review: Oppenheimer 1947). They have dealt with several different species, none of which has as yet been fully investigated and the results do more to whet one’s appetite than to provide a comprehensive account of the causal embryology of the whole group.
The earliest stage at which operations have been made is immediately after fertilisation. Such work goes back to the experiments of Morgan in the 1890s. The results of the most recent workers on Fundulus suggest that any one of the first two, or of the first four, blastomeres can give rise to a complete embryo. For example, Nicholas and Oppenheimer (1942) observed embryo formation in sixty-five cases out of seventy-two in which they had eliminated one of the first two blastomeres chosen at random. There seems to be little sign at this stage of an organisation centre which is localised as is the amphibian grey crescent. The existence of such a centre is, however, claimed by Tung and Tung (1944), using the eggs of the goldfish Carassius. They removed part of the uncleaved egg either by cutting it away with a knife or by pricking the future embryonic area and squeezing some of the cytoplasm out through the hole. They found that the earlier the operation was made after fertilisation, and the larger the part of the normal protoplasmic region remaining, the better was the differentiation. Fully normal embryos could be formed when the operation took place in about the first quarter of an hour after fertilisation and at least a half, or preferably more, of the protoplasmic region was left intact. In the abnormal and reduced embryos, mesodermal tissues sometimes differentiated rather well, but neural tissue does not appear to have been seen in the absence of accompanying mesoderm.
The same authors also divided the carly blastoderm into two parts, cutting along the cleavage planes in either the 2-, 4- or 8-cell stages. They found that each part might sometimes give rise to a complete and normal embryo. In other cases there was one normal embryo and one mass of cells with no histological differentiation; and in some cases both portions gave this undifierentiated result. Two complete embryos can also be fused together, in stages up to the 16-cell stage. They sometimes produced complete, separate, twins, but occasionally one well-formed but oversized embryo to which it was clear that both eggs had contributed. All these results are exceedingly similar to those which have been found in the Amphibia, and might indicate a similar underlying mechanism, namely an organisation centre which becomes located in the plane of bilateral symmetry soon after fertilisation, which is essential for the development of the axial structures of the embryo, and which is capable of considerable regulation, either when parts of it are removed or when two centres are fused together. But the critical evidence for such a centre is the fact that some blastomeres fail to give embryos. In view of the results of earlier work on Fundulus, it is perhaps unsafe to lay much stress on such essentially negative evidence.
In the stages immediately following this, a phenomenon has been noted which has sometimes been claimed to be a special feature of teleost development with no parallels in other groups. Oppenheimer (1934), 1936a) found that blastoderms of the minnow Fundulus, if removed from the yolk at the 16-cell stage or before and cultivated in salt solution, failed to gastrulate and developed only into featureless balls of cells. A rather similar result was reached for the goldfish by Tung, Chang and Tung (1945). They found that if eggs were divided latitudinally at the 1-cell and 2-cell stage, blastoderms could not develop properly unless they remained connected with more than half the total quantity of yolk. From the 4-cell stage the requirement of yolk was less and from the 8-cell stage onwards completely isolated blastoderms could develop into embryos. Devillers (1947) found that the blastoderm of the trout was unable to develop in isolation from the yolk even when it had been removed as late as the blastula stage. On the other hand, in the pike Esox similarly isolated blastoderms continued developing quite well.
The explanation of these facts is not clear. As a matter of fact a somewhat similar phenomenon has been reported in the Amphibia. Vintemberger (1936) showed that if, in the 8-cell stage of the frog, the four animal cells are isolated, they are usually unable to differentiate any axial organs although they contain part of the presumptive posterior region of the notochord rudiment. If however the four cells are placed on a base consisting of a mass of endoderm cells from the blastula, they differentiate very much better. There are several different ways in which these results and those in the teleosts could be interpreted.
(1) It is a fairly general observation that carly stages of embryos are more sensitive than older ones to the general injury produced by experimental operations. The increase in resistance usually continues at least until the main embryonic organs are laid down. It is noticeable, for instance, that it is easier to get good differentiation in tissue culture of chick blastoderms of primitive streak or later stages than of blastoderms taken from eggs in the first few hours of incubation. It may be that we are dealing merely with a general increasing ‘toughness’ of the embryos, which varies from species to species.
(2) Oppenheimer originally considered that the phenomena suggested the passage from the periblast into the blastoderm cells of some substance of an organiser-like nature which was necessary for the initiation of gastrulation (see 1947, where she is already somewhat cautious about this hypothesis). Tung, Chang and Tung considered their results supported this. The boundaries of the cells, particularly of the periblast but also of the blastoderm, are not very sharply marked in early cleavage stages of teleosts, and it seems quite possible that the blastoderm continues to incotporate further cytoplasmic material for some time after the cleavages have begun.
(3) It may be, however, that what the yolk provides is not some organiser-like substance but rather a relatively simple essential nutrient. This possibility is supported by the fact that Devillers (1949) found that blastoderms of the trout, which are unable to differentiate in pure salt solution, will do so when glucose or similar substances are added to the medium. Ina short note Trinkaus (1953) reports rather similar results with Fundulus.
Only if the second of these possibilities is the full explanation of the facts would we be confronted in the teleosts with a situation which differed radically from that in the Amphibia (since it is improbable that a similar process is the explanation of Vintemberger’s results). At present the matter must remain open.
Experiments on the gastrula stage show that, in teleosts, the invaginating chorda-mesoderm acts as an organiser in a manner extremely similar to that found in the Amphibia and in the chick. This was demonstrated almost simultaneously by Oppenheimer for the perch and Fundulus (19344, b, 1936b) and by Luther (1935, 1937) for the trout Salmo (Fig. 11.5). The parallel with the amphibian and bird organisers is extremely complete (see Review by Oppenheimer 1947). For instance, there is a similar type of regional determination by the different parts of the organisation centre, and evocation by dead tissues has been demonstrated.
It has been claimed (e.g. by Oppenheimer 1947) that the teleost ectoderm has some tendency to differentiate neural tissue without the intervention of the mesodermal organiser. Oppenheimer tends to interpret this in terms of an evocation by cytolysis, since in the abnormal embryos which gave evidence of such autonomous differentiation the cells were somewhat unhealthy. It appears however that the neural tissue was not completely without any accompanying mesoderm, but merely without accompanying differentiated chorda-mesoderm. The induction of neural tissue by mesoderm which does not include the chorda, and which may itself differentiate rather poorly, is well known both in the Amphibia and the chick, and it appears unnecessary to postulate anything more than this to explain the phenomena in the teleosts.
It appears probable that there is a considerable difference of a quantitative, if not of a qualitative, character between the teleosts and the Amphibia in the extent of regulation which can go on in portions of the blastula and gastrula separated from the main centre of the organiser region. Luther (1936) divided the pre-gastrulation blastoderm of the trout into four equal quadrants, each of which was then isolated by grafting it into the yolk-sac epithelium of an older embryo. All four quarters were found capable of differentiating into all the main embryonic organs, although these were usually somewhat chaotically arranged owing to the abnormal mechanical conditions. Soon after the beginning of gastrulation this equality between the four sectors disappeared. The sector diametrically opposite the position in which the embryo will form soon loses its power to differentiate axial organs. There develops, in fact, a well-marked gradient, according to which the capacity for differentiation falls off from each side of the main embryonic region. In Fundulus, Oppenheimer (1953) finds rather better differentiation of embryonic regions (three successful out of six) than of extra-embryonic germ-rings (five out of seventeen) but the figures are hardly sufficient to establish the existence of a gradient.
In a an early gastrula of the trout is opened, the tongue of invaginating material cut away and grafted into the opposite side of the blastoderm; b, surface view, showing the graft (dotted); ¢ and d, stages in the development of the host and induced embryos. (From Luther 193 5.)
In the Amphibia at similar stages (i.c. blastula and carly gastrula) the capacity of isolated ventral portions to regulate and form a complete embryo is, of course, very much less than that Just described for the teleost. It is however not completely absent, as Dalcq and Huang (1948) have shown (p. 177). The teleost situation is, however, more comparable to that in birds at the time of endoderm formation, as described by Lutz (p. 183); there, again, the centre at which the embryo will form is not sharply distinguished from the rest, and far reaching regulation is still possible.
Devillers (1951) has studied the orientation in which embryos develop from portions of the trout blastoderm left in place on the egg or rotated in various ways. His results indicate that, in spite of the power of regulation which is spread throughout the whole blastoderm, the future embryonic region has already some slight degree of dominance both in the blastula and still more the gastrula stages, and it tends to dictate the orientation of the embryo. This again is very similar to the situation in birds.
The well-known evolutionary changes affecting the head skeleton of the fish have been discussed from the epigenetic point of view by Devillers (1950).
Passing over the Amphibia, about which enough has already been said, we come in our survey of vertebrate types to the reptiles. They have been studied, recently, in the light of our newer knowledge of other groups, particularly by Peter (1938) and Pasteels (1936-7, 1940). The main points which require notice concern the formation of the endoderm and the invagination of the mesoderm.
The eggs are large and full of yolk; cleavage is partial; and we meet once more a blastodermal type of embryo. When the presumptive areas are mapped, it becomes clear that the make-up of the blastoderm is radically different from that of the teleosts. Instead of the presumptive endoderm and mesoderm lying round the margin of the blastoderm, they are located within it, the edge of the plate of cells being wholly ectodermal, There is considerable variation within the group in the mode of endoderm formation. In the Algerian turtle Clemmys leprosa, Pasteels finds that the blastoderm at the end of cleavage is single layered. At a point, which lies not at the edge of, but within, the blastodermic sheet, a groove appears and cells of the single layer become pushed inwards to form the endoderm, which thus owes its origin to a true invagination through a blastopore. In other species, such as the chameleon and certain lizards, the blastoderm never becomes single layered, and much, though perhaps not all, of the endoderm is formed in place by a delamination similar to that which was described above (p. 155) for the bird embryo.
After the blastoderm has become two layered, and endoderm formation is complete or nearly so, the invagination of mesoderm takes place through the same blastopore. The tissues move to the blastopore, sink down through it, and are pushed forward in the shape of a tube which extends towards the anterior. This used to be known as the ‘archenteric canal’, but since, according to the vital staining experiments of Pasteels, it contains no endoderm but is made up wholly of mesoderm, it should, he suggests, be called the ‘chorda-mesodermal canal’, Its roof will eventually become notochord, its sides somites, while its floor migrates further laterally to form the side-plates. At its anterior end the floor disappears at later stages, so that the canal leads from the blastoporal opening right through into the subgerminal cavity (Fig. 11.6).
Unfortunately, nothing whatever is known of the causal mechanisms of early reptilian development. The very slow differentiation of the eggs makes them seem likely to be difficult experimental material, and no one has yet had the opportunity or the boldness to tackle them.
Gastrulation in the turtle Clemmys. A, map of presumptive areas, before the invagination of the endoderm (shading as in Figure 11.3, dashed vertical lines, extra-embryonic). B, movements of surface tissues towards the blasto pore, from which the chorda-mesoderm canal extends. C, formation of endoderm from the blastopore at the beginning of gastrulation. D, the movements of tissues at a later stage through the blastopore into the chordamesoderm canal. (From data of Pasteels.)
For the sake of continuity, it is worth while repeating two points about the avian embryo in relation to what has just been said about the reptilian. In the first place, endoderm formation is probably by delamination, as in turtles, not by invagination from a definite blastopore. Secondly, while mesoderm invagination is beginning, streaming movements take place from the posterior towards the anterior; thus instead of the mesoderm being formed from a circumscribed blastopore which leads in to a long forwardly extending chorda-mesodermal canal, it originates from an clongated primitive streak, from the anterior end of which there juts forward only a short extension of invaginated material, namely the head process.
The major characteristic of the mammals (except for the most primitive group) is the adaptation of their embryos to intra-uterine life. Evolution has, in fact, been particularly active in the bringing about of changes in the early stages of development, and the group as a whole shows a very wide range of different conditions, which we shall only be able to sketch in very broad outline.
In the lowest group, the prototherian mammals or monotremes, such as echidna, the egg is still reptilian in gencral configuration, cleavage 1s partial, and a blastoderm is formed. Flynn and Hill (1939, 1942) find that endoderm formation takes place mainly by delamination, though there is also some movement of isolated cells out of the upper layer ito the deeper one.
In the higher groups (marsupials and true mammals), the form of the egg is completely different (Review: Nelsen 195 3). It is small, and contains little if any yolk. Cleavage is total. Endoderm and mesoderm formation, however, occur by processes which are clearly modifications of those seen in the reptilian ancestors. The cleavage gives rise at first to a rather solid mass of cells, but a cavity soon appears amongst them. This is excentrically placed, so that the embryo assumes the shape of a hollow sphere, or blastocyst, to the inner face of which a thicker cluster of cells adheres at one place (Fig. 11.7). This is the “nner cell mass’, and from it the main embryo will be formed. The remainder of the sphere is extra-embryonic ‘trophoblast’, concerned with anchoring the embryo to the wall of the uterus; it is probably to be compared with the outermost parts of the reptilian and bird blastoderms, the area opaca. From the inner cell mass, the endoderm probably forms by simple delamination, though little is known of this in detail and no vital marking experiments have yet been possible; there may well be some migration from the edges of the inner cell mass along the base of it towards the centre.
The mesoderm is formed from the outer surface of the inner cell masss. In this region the outermost layer of all, the “enveloping layer’ which is continuous with the main sphere of the blastocyst, either lifts away to leave a cavity (thus forming an amnion), or breaks down and disappears. The face of the inner cell mass thus exposed has in general an oval form in plan view, and on it there appears an elongated thickening, which is the primitive streak. In some forms, such as the rabbit, it is extremely like that seen in birds; in others, such as the mole and in man, the streak is shorter and there is a greater development of a chorda-mesodermal canal such as that found in reptiles. The invagination of the mesoderm has never been followed in detail by vital staining, but a few cinematograph films of rabbit blastoderms growing in vitro have been taken by Waddington, and a convergence of lateral material towards the streak from both sides has been seen; there is little doubt that the process is essentially similar to that of the bird and reptile. There are, of course, certain differences in detail. Thus in the rabbit the presumptive neural plate probably occupies a greater proportion of the embryonic area which corresponds to the area pellucida of the chick; and Waddington (1937) points out that the results of cultivation in vitro of posterior halves of rabbit embryonic areas suggests that there is less longitudinal movement up and down the streak than in birds.
Early development in a mammal. A, section of blastocyst, with inner cell mass. B, later stage, the main part of the blastocyst becoming the tropho blast (Tr), the lower layer of the inner cell mass becoming arranged as an epithelium, the endoderm. C, the inner cell mass arranged as two epithelia, ectoderm and endoderm; above the latter the original outer layer has be come elevated so as to form the amniotic cavity (R.m., ‘Rauber’s membrane’).
The first few steps have been taken towards a causal analysis of early mammalian development. Dalcq (19514, 1952), and several of his pupils (cf. Jones-Seaton 1950) are engaged in a detailed study of the differential staining and other properties of the cytoplasm of the oocyte and early developmental stages of the eggs of a number of species. Their most important results so far are, perhaps, the demonstration that there is some degree of bilaterality in the structure of the oocyte and unfertilised egg; and that the distinction between the embryonic and extra-embryonic regions arises gradually, and is not fixed at, for instance, the first cleavage division as has sometimes been suggested (Fig. 11.8).
The latter conclusion accords well with experimental work. This is, of course, technically very difficult in mammals. It is first necessary, to operate on the young stages, to remove the eggs from the mother, then to divest them of the tough jelly which surrounds them or to operate through it, and finally to return them to some situation in which they will continue their development. Rodent eggs, which are easy to obtain, seem very sensitive to the removal of their membranes, and are difficult to keep alive in any situation except the uterus, although they can be retransplanted back into another uterus with fair success (cf. Nicholas 1947, Willett 1953). The rabbit egg is in general tougher; the embryonic area at the primitive streak stage can be cultured in vitro for a time long enough for a fair amount of differentiation to occur. It is worth noting (cf. p. 231) that the older the embryo, the better it stands the conditions of artificial cultivation (Waddington 1937).
A little work has been done on the early cleavage stages. Nicholas and Hall (1942) succeeded in getting some development (up to just before the time the embryo makes its appearance) of one of the first two blastomeres, the other having been destroyed or removed. Pincus (1936) got rather similar results with the rabbit. Recently Seidel (19525) had been much more successful, One of the first two blastomeres of a rabbit egg was killed by pricking with a glass needle, without removing the jelly layer. The egg was then injected into the Fallopian tube of another rabbit at a suitable stage in the reproductive cycle, and fully normal young were born. The evidence that one blastomere was really killed seems quite convincing, and the operated egg differed genetically (in colour) from the host, so that there is no doubt that the young animal was derived from it and not from an uninjured egg of the foster-mother. Only two such animals have so far been described; one was fully normal, but the other showed defects on one side, rather similar to those which are found in newts’ eggs from half blastomeres when the first cleavage furrow does not divide the grey crescent region into equal parts. One may conclude that the rabbit egg (and presumably that of other mammals) is certainly not of the mosaic type, but may contain a localised organisation centre comparable to the amphibian grey crescent; this would probably be related to the bilateral structures described by Dalcq. (For experiments involving the alteration of chromosome numbers, induction of polyploidy etc. in mammals, see Beatty 1955.)
Three stages in the early development of the rat. Upper row, fertilised egg with pronuclei; middle row, four-cell; lower row, young blastocyst with inner cell mass. The eggs in the left column have been stained to exhibit alkaline phosphatase; those in the centre column RNA; and those in the right column mucopolysaccharides. (After original drawings of Dalcq.)
A few experiments have also been made at the primitive streak stage of rabbits although the material is technically very difficult to handle owing to its stickiness, transparency and resistance to cutting. Waddington (1937), failed to get any inductions by fragments of primitive streak transplanted between the epiblast and hypoblast in the manner used in the chick, but the material was certainly not extensive enough for this negative result to have much importance. On the other hand, the competence of the rabbit epiblast to react to neuralising stimuli was denmionstrated by cases in which such an effect was produced by grafts of chick primitive streak (Fig. 11.9); and rabbit primitive streak was also able to induce when grafted into the chick. It is highly probable, therefore, that the mamuinalian primitive streak is an organisation centre with functions similar.to those of the same structure in the bird embryo. It was also shown that development of the posterior region is possible in the absence of the anterior end of the streak (Hensen’s node), which some authors had considered to be an essential focus of embryo formation; again a result which parallels that in the bird. T6ré (1939) has reported the induction of neural tissue in the rat by grafts placed in the amniotic cavity, but his evidence, as published, is not very convincing, and it would be unexpected to find that tissues lying freely in such a cavity, in contact with the outer side of the ectoderm, could successfully induce; it seems more likely that he was dealing with embryos which were distorted as a result of the operations.
A small piece of chick primitive streak was grafted under the ectoderm of a rabbit embryo of the streak stage, which was then cultured in vitro. The graft has formed some neural tissue, and has induced a neural plate in the rabbit host. (After Waddington 1934.)
The results of direct operative experiments on mammal embryos have been supplemented by evidence of quite a different kind, which comes from an analysis by methods which have as yet been little used in other groups of animals, except the insects. A fairly considerable number of hereditary factors or genes are known which cause abnormalities in early developmental stages of mammals; perhaps their comparative frequency in this group is connected with the extremely rapid and radical changes which have led to the evolution of the group. The effects of these genes are often somewhat variable, and by a study of the variations it is sometimes possible to decide that one particular aspect of the abnormality is primary, and the remainder secondary consequences; from such arguments some insight into the causal sequences of epigenesis can be attained.
Dunn (1941) and in particular Gluecksohn-Schoenheimer (1949, 1953) working on the mouse, have made very important contributions in this way.
The first genes from which important embryological consequences could be deduced had their most obvious effects on the tail. The Brachyury gene T is a dominant, the heterozygous mice having short tails. The T/T homozygotes die before birth and in the embryos the whole posterior region of the body is missing (Chesley 1935). Detailed investigation showed that no notochord (or almost none) is ever formed in these homozygous embryos, and Chesley suggested that this was the primary action, the effect on other structures such as the nerve-cord being the result of secondary reactions of an inductive nature. This was confirmed, and the evidence made more convincing, when Gluecksohn-Schoenheimer investigated embryos of animals which were heterozygous both for T and for one or other of the genes f° and t1. These embryos have normallooking tails till an age of about eleven days, after which the tail becomes constricted at its base and degenerates, the young being quite tailless when born. Histological examination showed that even in the apparently normal tails of early stages, no notochord is present, although the neural tube, somites and tailgut are formed as usual. It seems then that the later degeneration is a consequence of the lack of the notochord, and some ‘inductive’ relation is indicated. It should be noted, however, that the relation is not quite that of evocation; even in the absence of the notochord, the neural tube is formed, presumably induced by the remainder of the mesoderm, but it is unable to persist. There is no exact parallel to this in other vertebrates, since the operative removal of the notochord, e.g. in Amphibia, does not lead to the regression of the neural tube, but only to a failure of its normal elongation and an alteration of its usual cross-sectional shape.
The homozygous f°/t1 embryos show more profound abnormalities. The whole endoderm tends to lift away from the rest of the inner cell mass some time before the beginnings of embryo formation are visible. The cells of the mass remain alive for some time, and some growth takes place, but there is no organisation of a primitive streak and no mesoderm appears; a day or two after the onset of the condition, the embryos die and are resorbed. Gluecksohn-Schoenheimer suggests that this may indicate that in the mammals, as in the birds, the endoderm may play an essential role in inducing the formation of mesoderm.
The same author has described a still more interesting gene, known as Kink. The heterozygotes show various spinal and tail abnormalities, and the homozygotes die at about nine days after fertilisation. Before death, a considerable amount of development has occurred, and the embryos exhibit a most remarkable range of conditions, most of which can be considered as twinnings or duplications of various kinds, ranging from almost complete and separate twins to doublings of the main axis, of the heart, the allantois, etc. There are also many examples of single more or less complete embryos accompanied by ball-shaped lumps of unorganised tissue. Gluecksohn-Schoenheimer compares the structures found to those which result from the partial or complete separation of the first two blastomeres in Amphibia. If this comparison is accepted, and it seems quite convincing, one would have to conclude, firstly that the mammal egg at an early stage is capable of profound regulation (a point already clear from the occurrence of identical twins and directly confirmed by the evidence from blastomere separation mentioned above [p. 238]); and secondly that it possesses an organisation centre which is, or becomes, localised as does the grey crescent in the Amphibia or the posterior part of the endoderm in birds. The most plausible explanation of the action of Kink is that it interferes with the gradual regionalisation within the egg, by which the organisation centre becomes focused on to the dorsal side. Unfortunately the evidence does not help us to decide at what stage in development this takes place in mammals. In the armadillo, in which four identical twins are normally produced from each egg, the initiation of the four rudiments appears to occur rather late, in the blastocyst stage (Patterson 1913), and although one would now like to see the newly fertilised eggs of this form re-investigated by the methods of Dalcg, it is perhaps likely that the mammals are like teleosts in that their dorsal plane is not finally fixed till a relatively late stage.
It may be mentioned that all the mouse genes just mentioned (T, #°, ft and Kink) are closely linked in the same chromosome; they may all be chromosome aberrations rather than true point mutations. Many other genes with generally similar effects (mostly not yet analysed embryologically) are known in the same chromosomal region. The causes for the association of this part of the chromosome with the primary organiser phenomena is quite unknown and present a very intriguing problem. Gluecksohn-Schoenheimer very tentatively suggests that possibly the spatial pattern of the genes in the nucleus may have some connection with the formation of the patterns of the early embryonic fields.
5. Comparative geography of the presumptive areas
In the past, attempts to compare the processes of gastrulation in the different classes of vertebrates have been made in terms of concepts which were derived from the static shapes which particular embryos may assume at certain points in their development. For instance, one tried to homologise the structure of the blastoderm of the bird before endoderm formation begins with the hollow sphere formed by the amphibian egg at the end of cleavage; or one enquired how the chorda-mesodermal canal in reptiles is related to the amphibian blastopore. Nowadays we regard gastrulation as a co-ordinated series of foldings and movements by which the various groups of cells which result from cleavage are arranged into the three fundamental layers of the ectoderm, mesoderm, endoderm. What ought to be compared is the complete set of movements in one form with the complete set in some other form, rather than any particular instantancous configurations which may be taken up during the process. The most manageable way of summarising the whole gastrulation of a particular group is provided by the map of presumptive areas at the late blastula stage, together with an indication of the directions in which these various areas will move.
When one looks at the maps of presumptive areas which have been described earlier, it is fairly easy to arrange them into a scheme in which they are brought into natural relations with one another. It is clear that such a scheme should start from a type of egg which is fairly small and has total cleavage, since the blastodermic forms of development must be secondary derivatives. Of the totally cleaving eggs, the most primitive fish (cyclostomes) and the Amphibia have maps which greatly resemble one another. The blastopore is placed low down on the vegetative side within an area of endoderm. Above this is a ring of presumptive mesoderm, which, certainly in the Amphibia and probably in the cyclostomes, extends right round the egg from the dorsal to the ventral side. Within this ring the presumptive axial mesoderm (notochord and somites) is concentrated somewhat towards the dorsal side, but extends much further laterally in the blastula than it will do after gastrulation is completed. The upper part of the egg is occupied by a cap of presumptive ectoderm, of which the region near the dorsal plane will become neural plate and the remainder epidermis. A very similar pattern is found in the protocordates (ascidians and Amphioxus), and we are probably safe in taking this as the basic type from which the others should be derived (Fig. 11.10).
There are two other main types to be fitted in. Between the cyclostomes and the Amphibia in the evolutionary sequence come the cartilaginous and bony fishes. These have a blastodermic type of development, and a characteristic feature of their presumptive map is that the margin of the blastoderm is made of presumptive endoderm or possibly mesoderm; the whole ectodermal area lies well inside the margin. The other groups—reptiles, birds and mammals—are evolutionarily more advanced than the Amphibia. Their embryos also develop from a blastoderm but the presumptive map differs radically from that of the fish in that the edge of the blastoderm is in this case constituted of ectoderm. The presumptive mesoderm lies inside the margin and when the endoderm occurs on the surface, as it does in certain reptiles, it is the most centrally placed area of all. As between reptiles, birds and mammals, there are obviously great similarities, such differences as are found being dependent on the extent to which the endoderm is formed by delamination or is originally at the surface, and on the degree to which the site of mesoderm invagination is drawn out into a primitive streak or concentrated into a chorda mesodermal canal.
Maps of presumptive areas. The large circles in the teleost and reptile maps represent yolk; other conventions as before. In the amphibian map, A represents the point at which the yolk would have to be inserted to give the teleost map, and B that required to give the reptile-bird map. (From Waddington 1952.)
The movements in the eggs of all the groups are rather similar in general type. There are movements of expansion in all directions, affecting particularly the ectoderm. There is a ‘dorsal convergence’, in which tissues become narrower from side to side and simultaneously elongated, and do this the more intensely the nearer they lie to the dorsal plane. Again, there are tendencies for invagination, that is, for certain tissues to plunge inwards from the surface and continue their movements at a lower level. Finally, in the blastodermal types, we find delamination, that is, a process by which an original single thick layer of tissue becomes rearranged into two thinner separate layers. Until we know more of the mechanical causation of the movements, it is not possible to make any further meaningful comparisons between them.
The main aspect of the comparative scheme which one would like to understand more fully is the relation between the two sorts of blastodermal development. Two main ways of regarding this have been proposed. Dalcq (1938) and Pasteels (1940), who were the first to discuss the matter at all fully, were content to accept an irreconcilable duality within the evolutionary system of the vertebrates. According to their scheme, the teleost map should be regarded as derived from that of the Amphibia by the insertion of a large mass of yolk near the vegetative pole, while the bird-reptile map is derived by inserting the mass of yolk somewhere within the area of presumptive epidermis (at point Bin Fig. 11.10). This is the simplest and most straightforward scheme, but the idea of such drastic qualitative differences of egg structure within the vertebrates is not entirely satisfactory.
Waddington (1952b) has suggested that one ought to take into account not only the disposition of the presumptive areas on the egg surface but also their arrangement in depth. The blastodermal types might be derived from the amphibian arrangement, not by opening the latter at some point and spreading it out so that it can sit as a cap on top of a large mass of yolk which is inserted in the hole, but rather by imagining that the amphibian blastula becomes yolk-free, and then that the whole sphere is squashed down on to the surface of the separated yolk mass (Fig. 11.11). If, when the spherical blastula is distorted in this way, the line along which it folds (the ‘primitive edge’) lies wholly within the presumptive mesoderm, then the blastoderm will have a mesodermal margin and the whole of the endoderm will already be beneath the surface before gastrulation begins. If the primitive edge on the dorsal side lies a little lower, within the endodermal region, some of the dorsal margin of the blastoderm will be endodermal. According to the exact position of the line, one can easily arrive at the conditions seen in the Selachia or teleosts. Again, if the primitive edge lies higher on the amphibian map, it will, particularly on the ventral side, cut into the presumptive epidermis and the blastoderm will have an epidermal margin in that region.
In order to explain the fact that the whole of the Bieeodeaeel margin in birds is ectodermal, we should have to postulate an expansion of the presumptive epidermal area in addition to the flattening of the spherical blastula. This makes the scheme more complex than that of Dalcq and Pasteels. Its main merit is that it gives an example of one way in which it is possible to envisage both the fish and bird blastoderms as two modalities of one general process; and further, it draws attention to the need to consider the disposition of the presumptive areas in depth as well as on the surface. It is most desirable that we should find out more about the location of the presumptive areas in the extremely yolky types of amphibian eggs, particularly those of the Gymnophiona. In these, much of the endoderm seems to be beneath the surface before gastrulation begins and to become distinct by a process of delamination, which would seem to accord better with Waddington’s scheme than with the Dalcq-Pastecls’ one. Gastrulation in these eggs has, however, not been studied since Brauer’s work in 1897.
A B FIGURE II.1I
A, the basic vertebrate map, with a line of folding (‘the primitive edge’) indicated by a broken line. B, the blastula squashed down on to a mass of yolk, having folded along the primitive edge. If such a deformation is to produce the bird map, there would also have to be an expansion of the epidermal area. (From Waddington 1952.)
6. Comparative causal embryology of vertebrates
The various types of vertebrate eggs, different in many respects and yet all anatomically related in the ways we have just discussed, provide a wonderful opportunity for studies on the kind of alterations in epigenetic mechanisms which evolution can bring about. There is no doubt that one ought eventually to envisage evolution not as a process which merely causes modifications in the structure of adult animals, but rather as one by which the causal sequences of development have been changed. These changes have affected even the basic mechanisms which operate in the early stages of development. Dalcq (19510) has referred to mutations which affect these stages as ‘onto-mutations’. Little is known in detail about them from the genetical side, though many of the factors which are labelled (and usually dismissed) as ‘female-steriles’ probably fall into this category (cf. p. 135). The comparative study of the epigenetic systems in different groups of vertebrates gives an opportunity to see the kinds of effects which such gene changes produce.
The first noteworthy epigenetic event in the development of vertebrates is the fixing of the plane of bilateral symmetry. In the Amphibia, as we have seen, the future organisation centre normally becomes located soon after fertilisation and is sometimes visible as the grey crescent. In the other primitive non-yolky type of vertebrate egg, that of the cyclostomes, Montalenti and Maccagno claim that the situation is very similar. In the protocordates (ascidians and Amphioxus) the dorso-ventral plane is also fixed definitely at an early stage. In both the ascidians and amphibians there is indeed some evidence that the dorso-ventral plane is at least foreshadowed before fertilisation occurs. In the latter the position of the plane is at first labile and only gradually becomes definitely determined. By the time cleavage is completed and the blastula stage reached, the dorsal plane is firmly determined and it is difficult to cause a new dorsal region to appear elsewhere when the egg is divided in two. Such powers of regulation are, however, not completely extinguished until considerably later (p. 177). The results obtained by Bytinski-Salz on the cyclostomes suggest that the same is true in that group.
In the blastoderms of both the teleosts and the birds the situation seems to be rather different. We have some evidence (Vakaet 1953) that the growing oocyte of the teleost has a bilateral structure, but in the developing embryos the position of the dorso-ventral plane is quite easily alterable until a late stage of the blastula, as is shown by the results of Luther in teleosts and Lutz in birds. It seems probable that the original bilaterality in the oocyte is swamped by the great flood of yolk which is laid down, and that a bilateral structure is only gradually and slowly re-established. It is true that Tung and Tung (1944) argued that, in the goldfish, an organiser-like region beomes located on one side of the egg shortly after fertilisation, and thus endows it with a bilateral symmetry, but it is doubtful if their experimental material was adequate to sustain this conclusion (p. 230). It seems likely therefore that we have to accept a difference in timing as between the holoblastic and blastodermal eggs, the dorsoventral plane being determined much later in the latter.
In the Amphibia the agent which determines the dorso-ventral plane was the inner yolk mass. In the teleosts it seems to be the periblast. It 1s difficult to say how far these two can be considered as in any way comparable, either in their anatomical derivation or their mode of action. In both groups what is determined is, in the first place, the invagination of endoderm; and hard on the heels of this comes the mesoderm, which proceeds to be invaginated around the same focus. In birds we have no direct evidence as to the mechanisms which determine the posterior end of the endoderm, which is the region that takes the lead as soon as the dorso-ventral plane is fixed. In this form, however, another stage is interpolated into the normal sequence, since there is something of a pause between endoderm formation and the start of invagination of mesoderm at the primitive streak, and we find that the endoderm exerts a causal influence which determines the site of mesoderm invagination. The apparently straightforward, follow-my-leader behaviour of mesoderm towards endoderm in the lower groups has, therefore, been expanded in the birds into a cause-effect sequence.
We still know too little about the epigenetics of mammals to fit them with any confidence into the scheme. There is evidence (Dalcq, p. 238) that the oocyte and newly fertilised egg have some degree of bilaterality of structure. Seidel, as a result of his separation of blastomeres, argued that there is a localised organisation centre. This result, however, was based on only two sets of operations and is exceedingly tentative. On the other hand, the evidence suggests that the formation of identical twins may occur at much later stages; and on the whole it secms probable that in mammals, as in birds, the final determination of the dorso-ventral plane occurs rather late.
It seems probable that, in all the vertebrates without exception, determination of the dorso-ventral plane fixcs, in the first place, the location of the endoderm and, secondly, that of the mesoderm, but that the ectoderm remains quite indifferent till it is acted upon by an organising influence coming from the mesoderm. Grafting experiments have given direct proof of the inducing power of the presumptive mesoderm in cyclostomes, teleosts, Amphibia and birds and the demonstration is only slightly less clear-cut in mammals. In all groups there is evidence that at the time the mesoderm starts to be invaginated the fate of particular regions of it can still easily be altered, but that, as invagination proceeds, it is affected by a process of regionalisation so that different arcas become determined, not only as to the tissue into which they will develop (as notochord, somites, nephros, etc.), but also as to their position on the anterior-posterior axis (brain, spinal cord, tail, etc.). There is some not very compelling evidence (p. 187) that in birds the presumptive forebrain has a tendency to develop into neural tissue independently of any induction by mesoderm. If this is so it may be connected with the fact that, in this group, the determination of the site of mesoderm formation (the primitive streak) is a comparatively long-drawn-out process caused by the endoderm. In other groups the development of neural tissue seems to be completely dependent on induction, and the whole responsibility for the appearance of the main embryonic axis can be attributed to this process and to the self-individuation (i.e. regionalisation) of the mesoderm.
It seems therefore that all the groups of vertebrates employ essentially similar epigenetic mechanisms. The main changes which have occurred within the group appear to be: (1) The swamping of the initial structure of the oocyte by the enormous quantities of yolk laid down in fish and birds, and a consequent postponement of the time at which the plane of bilateral symmetry is finally determined, and (2) the interpolation of anew causal relationship between endoderm and mesoderm in birds (and possibly in reptiles and mammals) in consequence of the difference in the time of invagination of the two layers.
Needham 1942, pp. 320-30; Dalcq 1938, pp. 7-58. For comparative gastrulation, Pasteels 1940, Waddington 1952b or 1952a, pp. 39-50. For teleosts, Oppenheimer 1947; for mammals Gluecksohn-Schoenheimer 1949, Dalcq 19514.
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