CHAPTER X THE EPIGENETICS OF THE EMBRYONIC AXIS
The account which has so far been given of the development of amphibian and bird embryos has been concerned almost entirely to describe the events which take place; it is time now to see how far we can go in giving a causal explanation of them. Attempts to analyse experimentally the processes which bring about developmental change have of course been made for many years, but it is only in the last three or four decades that we have begun to understand the causal connections involved. This deepening of our understanding may not too unfairly be dated from Spemann’s discovery in 1918 of the ‘organiser’, which will be discussed in some detail below. There were, of course, foreshadowings in earlier years of Spemann’s discoveries, as there always are of important scientific advances; but in this case they were so slight, and their importance so little understood even by their authors, that they serve rather to illuminate the magnitude of Spemann’s advance than to dim its lustre. It is therefore of great importance to understand exactly what Spemann discovered and the way in which it is significant (Reviews: Spemann 1938, Dalcq 1941, Needham 1942, Lehmann 1945).
Since most eggs are small, the manipulative difficulty of experimenting on them is considerable, and the older experimental embryologists had found themselves restricted almost entirely to the expedient of cutting the egg into fragments, which were then allowed to develop in isolation. The result of such an experiment was normally either that the fragment developed into the fate which would have been in prospect for it if it had remained untouched in the egg, or that it developed into a complete embryo. In the former case, the egg was called a mosaic egg, in the latter a regulation egg; and, if a further step in theoretical analysis were called for, the fragments of the former type might be called ‘unipotent’ and those of the latter ‘totipotent’, these words implying that the former had only one potency for development while the latter had all the potencies required to produce a complete organism. Where it was possible to perform experiments on a series of younger and older stages, it was commonly found that while fragments from an carly stage might be totipotent, those from a later one had become unipotent.
There grew up a considerable body of discussion of the way in which the
173 174 PRINCIPLES OF EMBRYOLOGY
transition took place. For instance the American embryologist Lillie (1929) spoke of a process of ‘segregation’ or ‘differential dichotomy’ by which the totipotence of the original egg was sorted out into a set of unipotencies distributed to the appropriate parts of the embryo. But although the words totipotent and unipotent may be quite convenient additions to the embryological vocabulary, it is a mistake to allow their seductive technical flavour to conceal the fact that they suggest no explanation of anything. Moliére many years ago made fun of the doctors who thought they could explain the sleep-producing action of a drug by attributing to it a ‘soporific quality’; and a “developmental potency’ is a phrase of the same kind. To say that a certain part of an egg has a potency for neural tube formation, for example, means no more than that it has been observed in certain circumstances to become neural tube; and any possibility of providing a causal explanation of the phenomenon lies not in the invocation of potency, but in analysing the conditions under which such a development occurs. Spemann’s service was to discover phenomena which allow one to pass beyond such tautological concepts as potency, and take the first step in identifying the causal interactions involved in development. Naturally the revelation of the first step immediately prompts new questions as to the steps beyond; but in science, as in much else, c'est le premier pas qui compte.
Spemann’s success was partly due to a wise choice of experimental material. The newt’s egg is large, and lends itself to the easy performance of grafting and cutting experiments. In the early years of the century Spemann cut the egg in half at various stages from fertilisation onwards, and showed that each half might produce a complete embryo when the operation was made at any stage before gastrulation. During gastrulation, the ‘totipotence’ of the halves was rapidly reduced, and by the end of it each half gave rise to only a half embryo, whatever the plane in which the cut was made. The crucial problem was therefore to discover what happened during gastrulation to ‘restrict the potencies’ of the parts.
A clue was present in the fact that although in some experiments both halves of an early stage gave rise to complete embryos, in others one developed completely while the other formed only a mass of cells lacking any sign of the organs of the embryonic axis. We have seen earlier (p. 148) that in fact only those halves containing the grey crescent material develop properly; but in the newt the grey crescent is not clearly to be seen, and the location of the important region was therefore not obvious. Spemann ran it to earth by a series of experiments on the early gastrula. He found that if he separated dorsal and ventral halves, only the dorsal ones developed an embryo; again, if he cut the gastrula in half along the equator, THE EPIGENETICS OF THE EMBRYONIC AXIS 175
only the vegetative half developed; thus the crucia] region is in the dorsal vegetative quadrant which contains the blastopore. The next step was to graft a fragment from the blastopore region into another location in the egg. It was found to develop, whatever its new position, into part of an embryonic axis (Figs. 10.1, 10.2). Grafts from the presumptive ectoderm,
An organiser graft by the “Einsteck’ technique. A piece of tissue from the
neighbourhood of the blastopore of one gastrula is inserted into the blasto coel of a second gastrula. The movements of gastrulation press it against the
ventral ectoderm of the host, in which it induces a secondary axis. The
diagrammatic view of this (below) shows that the organs may be normal
in shape, although formed partly from the graft (black) and partly from host tissues. (From Holtfreter 1951.)
on the other hand, did not behave in any uniform manner, but developed in accordance with their new surroundings.
This not only showed that the blastopore region is the part which is essential for the formation of an embryonic axis, but also suggested that it acts as it were as a focus around which the whole egg is organised. Spemann suggested that when a graft of ectoderm develops similarly to its new surroundings, it is really its new relationship to the blastopore which is determinative. A final proof of this came a few years later, when Spemann and Hilde Mangold (1924) made grafts of the blastopore region of gastrulae of Triton alpestris into gastrulae of another species of newt, T. taeniatus. The tissues of the two species can be distinguished in stained 176 PRINCIPLES OF EMBRYOLOGY
and sectioned material and this made it possible to demonstrate conclusively that the grafted tissues had not only themselves developed into parts of axial organs, but had also caused the surrounding host tissue to do so, although its presumptive fate was to become mere epidermis.
FIGURE 10.2 1. View of vegetative pole of a newt gastrula. The main blastopore is to~ wards the bottom, and a second blastopore region, from another gastrula which was vitally stained with Neutral Red, has been grafted into the ven tral side of the vegetative region. 2, 3. Stages in the invagination of the normal and grafted blastopores. 4. The grafted material has become completely invaginated and disappeared below the surface. It has induced a neural plate (ind. #1.p.). One of the normal neural folds of the host embryo can be seen (h.n.p.).
(Original, from a time-lapse cine film.)
Such a reaction was spoken of as an embryonic induction, and the region near the blastopore from which inducing grafts can be obtained was called by Spemann the organisation centre or organiser of the embryo. The discovery of the organiser gave embryologists for the first time the power to control the direction in which an embryonic tissue develops, THE EPIGENETICS OF THE EMBRYONIC AXIS 177
and to do this by means of a mechanism which operates during normal development. Thus a piece of gastrula ectoderm can be made to differentiate into neural tissue by placing it in the near neighbourhood of an organiser. This remains a step of the utmost importance in the history of embryology. It did not, of course, provide a final answer to all the problems of development. Some authors have been so zealous in emphasising this (e.g. Weiss 1935, 1939, 19500) that they have tended to suggest that the whole concept can be dropped from our thinking, which is indeed to throw the baby out with the bath water. What is called for is not a rejection of Spemann’s ideas, but a further analysis and clarification of them. The phenomena which he had revealed are certainly complex. Thus ‘induction’ has two different aspects, evocation and individuation, while an essential role in the whole process is played by the ‘competence’ of the reacting tissues. We shall have to discuss these concepts further below.
The next few years following the discovery of embryonic induction were naturally spent in a general survey of the organiser’s properties. The extent of the organisation centre was examined by inserting small fragments of one gastrula into the blastocoel cavity of another; the invaginating mesoderm of the host presses the graft up against the ectoderm on the ventral side, and if it possesses any inducing power, anew embryonic axis appears there (Fig. 10.4, p. 180). It was found that the organiser is at least as large as the region which will develop into the axial mesoderm, (the notochord and somites); that is, the original grey crescent. But its boundaries are somewhat vague, since the inducing power falls off gradually from the centre of this region. In early stages, some degree of inducing power has been shown to exist even in ventral regions (Dalcq and Huang 1948, Dalcq 1950); and the capacity for induction is quite definite though weak, outside the axial mesoderm in late gastrulae, so that even the lateral parts of the mesoderm can induce when planted into quite young hosts (Waddington 19362). The organiser region is, in fact, a ‘field system’ in which the peripheral parts are dominated by the centre.
Fragments of early gastrulae which have the power to induce always themselves develop into some mesodermal tissues, although they may also form neural, and even endodermal tissues. This point was very well investigated after Holtfreter worked out a salt solution in which embryonic amphibian tissues would survive and develop, using up their stores of yolk as nutrients. He showed (1938a) that isolated pieces of presumptive mesoderm could develop into very many different tissues, although the region from which a given organ was obtained was roughly centred on 178 PRINCIPLES OF EMBRYOLOGY
that from which it would normally develop (Fig. 10.3). The remainder of the egg had much less inherent capacity. In particular, the presumptive neural plate and the presumptive epidermis were alike in that when
The urodele gastrula seen from the side; showing A, the presumptive fate
of the areas (what they will normally develop into); B, what they will self differentiate into when small parts are isolated in saline; C, the “prospective
potency’, i.e. what they can be induced to develop into; D, the region of
the primary organisation centre (‘head’ organiser, dots; ‘tail’ organiser, dashes). (From Holtfreter.)
isolated they both formed merely a generalised epidermal tissue with no special differentiations. This region of the gastrula must not, however, be considered as completely neutral and characterless. It has one most important property; namely the readiness to react to the organiser stimulus. This property shows no sign of being in existence before the onset of THE EPIGENETICS OF THE EMBRYONIC AXIS 179
gastrulation, and is certainly no longer present by the end of it; organiser grafts into old gastrulae (closed yolk-plug stage) no longer produce inductions. During the stage when the gastrula ectoderm can react, it is said to be competent (Waddington 1932), and the period when the reaction is possible is the period of competence. These words are also used of other tissues which, at later stages, become competent to react to the organising stimuli, which, as we shall see, are exerted by the various organs as they gradually develop (Chapter XII).
Competence can be thought of as a state of unstable equilibrium; the tissue is poised between two or more alternative paths of development, and may follow one or the other according to the organiser stimuli acting on it. In the case we are now discussing the most obvious alternatives are between the epidermal development path or the neural one. But actually there is a third; the presumptive ectoderm may be converted into mesoderm. This may occur even when the organiser graft is made merely by inserting a fragment into the blastocoel, but it is better shown by grafting a small fragment of presumptive ectoderm into the middle of the presumptive mesoderm just above the blastopore lip; it is then found that the graft becomes invaginated along with the host mesoderm, and takes part in the formation of the host’s mesodermal organs (Spemann and Geinitz 1927, Raven 1938). Further, if such a graft, after being allowed to invaginate, is then removed and grafted into still another gastrula, it has now become an organiser itself, and can perform an induction in its new host. We shall return later when discussing the physiology of induction, to this ‘infectivity’ of the organiser (p. 195).
By the end of gastrulation, the action of the primary organiser is over. The competent tissue has been definitely swung into one or other of its possible types of development; it is now definitely started on the way to becoming either neural tissue, or epidermis, or mesoderm, Within each of these types, a good deal of latitude is still open to it; it is still not finally settled whether it will become brain or spinal column; skin or an car vesicle or a lens; muscle or mesenchyme or part of the urinary apparatus. But the initial choice of path has been made. One step of development has been, in the usual phrase, ‘determined’, and if the tissue is allowed to develop at all, it will develop in accordance with that determination.
We have so far discussed the action of the organiser in terms of its effect on tissue differentiation, speaking of its results as the formation of neural tissue or epidermis, etc. This is a simplification of what actually happens. The result of an organiser graft is often the production of an induced organ, i.e. something in which the tissues are shaped into a more or less definite structure and related to one another as they would be in a part 180 PRINCIPLES OF EMBRYOLOGY
of a normal embryo. In these organs, parts derived from the graft are often closely intermingled with others induced from the host, both together forming a more or less unitary structure. In such cases, the organiser has done something more than merely throw the host tissues into a certain developmental pathway; it must have specified in detail the particular structures, and parts of structures, which the competent tissues form. Spemann (1931, 1938) followed his original discovery of the organiser phenomenon by showing that different regions of the organisation centre have different properties in this respect (Fig. 10.4). The presumptive anterior regions tend to induce head structures and the pre-" sumptive tail regions tend to induce tails. There is thus a regional differentiation within the organisation centre, and the regional properties of a given part can be transmitted to the competent tissues in contact with it. In the early gastrula, the regional structure, although definitely present, is not yet firmly fixed. In the first place, although an isolated piece of the
Head and tail organisers in the newt. 1, A fragment of tissue (presumptive
anterior mesoderm) is taken from near the dorsal lip of an early blastopore
and placed in the blastocoel of another embryo so as to arrive at the anterior
(1) or the posterior (1b) region of the host. In either case it induces a head.
2, A similar piece taken from a late yolk-plug gastrula may fail to induce a head (2b) unless it is near the host’s head (2a). THE EPIGENETICS OF THE EMBRYONIC AXIS 181
organiser has a tendency to form some particular organs of the embryonic mesoderm, it usually develops into, and induces, a larger region of the embryo than it would have done if left in place. Moreover, the different regionalities can influence one another. Tail organisers, grafted into the head region of a host, often induce heads instead of tails. This is not because the competent ectoderm there has an inherent tendency to react by forming a head; it is a consequence of the influence of the nearby host head organiser which tends to force the small grafted fragment to take on the character of the part of the embryo in which it lies. Again, if a small part of the organisation centre is excised and replaced in reversed orientation its surroundings may force it to conform with them, so that a normal embryo results (Abercrombie 1950 in the chick, Waddington and Yao 1950 in Amphibia, see p. 458).
The action of a graft in forming, together with what it succeeds in inducing, a more or less complete organ, and the action of one organiser on the regionality of another are both examples of a tendency by a fragment of organiser to form a whole and complete unit—cither a complete organ or a complete embryo. Spemann at first tended to think that this unit-forming tendency was an essential property of the organiser; in fact, the very name ‘organisation centre’ which he gave his discovery seems to imply something of that kind. However, Waddington and Schmidt (1933) were able to show that this is not the case. The capacity to perform an induction of some kind or another can be dissociated from any tendency to produce the missing parts of a complete unit, or to induce any specific region of the embryo. The two aspects of organiser action can be experimentally separated from one another, and one can have grafts which induce but which cannot truly be said to organise. The first clear demonstration of this arose during experiments on the organisers of the chick embryo, and it is now time to turn to a consideration of the epigenetic features of bird development.
The study of the epigenetic processes involved in avian development (Review: Waddington 1952a) was held up by technical difficulties greater than those offered by the Amphibia. There were not only the usual obstacles of small size, but the embryo is located under a hard shell and viscous albumen, and on top of a fluid ‘yolk’. Early experimenters, such as Hoadley, succeeded in cutting the blastoderm in half and following the development of each part; and others, particularly Willier and his students, cut out small fragments of the embryo and got them to develop in isolation by placing them where they could obtain nourishment from 182 PRINCIPLES OF EMBRYOLOGY
the blood supply of the chorio-allantoic membrane of much older chick embryos. Neither of these methods enabled one to investigate the effect of one part of the embryo on its neighbours, which Spemann had showed to be of fundamental importance in the Amphibia. And, owing to the way the blastoderm is built up of three superposed and closely adherent Jayers, even the isolated fragments contained a not-well-defined mixture of tissues and were not comparable to the specific gastrula pieces cultivated by Holtfreter in his salt solutions. As a result, very few definite conclusions could be drawn from work which relied on these techniques. Such theories as were suggested were cast in the old terms of ‘potencies’ and ‘embryonic segregation’.
New possibilities were opened up when the technique of tissue culture was adapted to the task of keeping alive the entire blastoderm after removing it from the egg and cleaning it of the adhering albumen and yolk. In such blastoderms, the three germ-layers can be separated from one another, at least in certain regions, and fragments can be grafted into abnormal places where their influence on the surrounding tissues can be studied (Waddington 1932).
We have seen that the morphological changes going on during gastrulation are more complex in the birds than in the Amphibia, and so are the organiser phenomena, probably because the morphological and physiological processes are intimately connected. The first stage in bird gastrulation is the formation of the endoderm, and this is the earliest stage at which an experimental attack has proved possible. In the young blastoderm, in which the primitive streak is just beginning to be indicated, the endoderm may be peeled off, rotated about a vertical axis, and replaced either head to tail, or so that its longitudinal axis makes a right-angle with that of the epiblast (Waddington 19332). It is found that such a rotation has a powerful influence on the elongation of the primitive streak and of the embryo which eventually develops. If the rotation has been through a right-angle, the head end of the embryo is curved round towards the new position in which the head end of the endoderm has been placed. With a complete head to tail rotation the usual result is that the embryo is greatly shortened, and does not extend fully across the area pellucida. It is clear that the elongation of the epiblastic part of the embryo tends to proceed in the posterior-to-anterior direction of the endoderm. In some cases of head-to-tail rotation the influence of the endoderm is more far reaching, and a new primitive streak and embryo are induced, running from the posterior part of the endoderm to meet the original embryo head-on in the centre of the area pellucida. The endoderm is therefore in some sense an organiser, since it can call forth the developTHE EPIGENETICS OF THE EMBRYONIC AXIS 183
ment of a new embryo. But we do not know how far it can be said to determine the developmental fate of the epiblast; probably it only determines that a new primitive streak shall be formed, leaving the later events undecided. As we shall see, a second organiser action has to go on within the streak before the definitive embryo appears. The organising action of the endoderm is perhaps connected with the posterior-anterior streaming which it seems to undergo itself (Fig. 10.5).
On the left is a chick embryo, cultured in vitro, in which the anteriorposterior axis of the endoderm was reversed in the early streak stage. Two embryos have developed, one (with tail end towards the bottom of the page) in the original direction of the epiblast, the other in the opposite direction; the latter must have been induced by the endoderm. (From Waddington 1933.) On the right the small diagram shows a blastoderm of the duck, at the time of laying, transected in the dorso-ventral plane; twin embryos develop. After Lutz 1949.)
The early endoderm, in the stages when the primitive streak has not yet appeared, is in a very labile condition. Lutz (1949) has shown that if a duck’s blastoderm is cut across at this period, both parts may form a complete embryo, and this is true whatever the orientation of the line of section (Fig. 10.5). The endoderm, then, can regulate very well and its inducing capacity is present, in some degree, throughout the whole of it. If one studies the orientation of the embryos which are formed in this way, it is found that that developed in the posterior part of the blastoderm
N 184 PRINCIPLES OF EMBRYOLOGY
always retains its original anterior-posterior axis, whereas the polarity of other regions is more labile. This is an indication that the posterior region is the dominant part of the ‘endoderm-field’ (Lutz 1952).
There is not much difficulty in making grafts of pieces of blastoderms of the primitive streak stage, once the method of growing the embryo in tissue culture has been adopted. But the procedure differs slightly from that usual in the Amphibia. In the latter, a hole can be cut in the gastrula, and a graft inserted so that it lies flat with the main surface of the egg. If a similar operation is made in the chick, the edges of the wound often curl back, and fail to heal up with the edges of the inserted graft. It is therefore more effective in bird embryos if the epiblast and endoderm are slightly separated, a pocket formed between them, and the graft inserted into it; this corresponds, more or less, to the method of pushing a graft through the roof of the amphibian gastrula into the blastocoel.
The only other important difficulty which arises in the chick is connected with the interpretation of the results. We saw that in the Amphibia the conclusive proof of an inducing action by the grafted organiser was given by making the graft of a different species from the host, so that the two sets of tissues could be distinguished in sections, and convincing evidence produced that parts of the secondary embryonic axis had been formed from host tissues. In birds, it has so far been impossible to find two species whose embryonic tissues can be distinguished with certainty; Waddington and Schmidt (1933) made many grafts between chick and duck embryos and obtained structures which were certainly inductions, but the tissues were similar in appearance, and host and graft could only be recognised in a general way, by the fact that the graft tended to remain a rather separate lump. The final proof of the reality of induction had to be obtained by a different method. This was done by taking two blastoderms, removing the endoderm from each of them, then placing them with the mesoderm faces together, and with the two primitive streaks not on top of one another. When the whole of such a combination is erown in vitro, as many as four embryonic axes may appear; one from each of the original primitive streaks, and two more, one induced by each streak in the other epiblast against which it lies (Waddington 1932). The neural grooves are still firmly attached to the epiblast from which they arose, and it can be quite definitely seen that the secondary ones have been snduced and were not formed from the original streaks. The origins of the mesodermal parts of the embryonic axes is not so certain, and one must guess that they are formed partly from the original streak and partly by induction.
The organising powers of the different parts of the streak can be THE EPIGENETICS OF THE EMBRYONIC AXIS 185
investigated by inserting small fragments between epiblast and endoderm, in the way described above (Figs. 10.6, 10.7). As in the Amphibia, the inducing region turns out to be about co-extensive with the presumptive axial mesoderm. In birds, this is a fairly small part of the whole mesoderm, since there is a large proportion of lateral mesoderm destined to migrate
Above, on the left, a chick blastoderm of the primitive streak stage; at its
right anterior a piece of streak from another blastoderm has been grafted
between the epiblast and endoderm. On the right, part of a host (duck)
blastoderm, with to the right a secondary head region induced by the anter ior half of a chick primitive streak. Below, a section showing the induced
brain (to the left) underlain by the graft, which has also developed some neural tissue. (After Waddington and Schmidt 1933.)
out to the sides of the blastoderm; this material probably has a wea inducing capacity, and thus, in the fully grown primitive streak stage, grafts from the posterior half of that structure, consisting entirely of the lateral mesoderm, may sometimes succeed in inducing, though they often fail (Abercrombie 1954). There is also some organising capacity in the regions of the blastoderm just lateral to the streak (Abercrombie and Bellairs 1954). 186 PRINCIPLES OF EMBRYOLOGY
In birds it has not been possible to make any experiments which truly parallel Holtfreter’s isolations of gastrula fragments into salt solutions. For one thing, the bird tissues do not contain enough yolk to continue living entirely on their own; they have to be supplied with nutrients, either from the tissue-culture medium or some form of blood supply, and it cannot be assumed that such media are as neutral in effect as is a salt
On the left of the host’s axis, an induction has been produced by the poster ior half of the same streak used in the embryo shown in Figure 10.6, On the
right are three sections, showing how the induced axis (on the right in the
figures) has combined with the host axis. (After Waddington and Schmidt 1933-)
solution. Further, the mesoderm and ectoderm adhere closely together in the epiblast, and it is next door to impossible to obtain fragments which contain nothing but presumptive neural tissue. Extensive isolation work has, however, been carried out, chiefly by American embryologists who used fragments of epiblast containing both ectoderm and mesoderm (and often endoderm as well), which were grafted on to the chorio-allantoic membrane of older chick embryos, where they become vascularised and continue their histological, though not their morphological, differentiation. The most important result they have obtained is to show that in the THE EPIGENETICS OF THE EMBRYONIC AXIS 187
primitive streak there are rather vaguely delimited areas which exhibit tendencies to produce specific organs, such as heart, eye, liver, etc. (Reviews: Rudnick 1944, 1948). It is probably safe to assume that these rough localisations are, in the first place, characteristics of the mesoderm, and that a tendency to forma definite ectodermal organ such as the cye indicates the localisation of a mesodermal eye inductor rather than of the eye itself (Fig. 10.8). If that is so, the phenomena are essentially similar to the
© Tr 30 /Fore-brain| € + Eyes, Gd Mid-brain/ | i © Hind-brain fe TT
aLiver @Heart ©Chorda 4Thyroid ©Nephros
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(2) Map showing regions of the definitive primitive streak blastoderm from which various tissues differentiate in chorio-allantoic grafts; ectodermal tissues on left, mesodermal on right. (After Rudnick 1948.)
(b) Regions of the head process blastoderm from which heart muscle differentiates in chorio-allantoic grafts; the closeness of the hatching indicates the relative frequence of heart differentiation. (After Rawles 1943.)
vague localisations of different capacities within the amphibian mesoderm described by Holtfreter (p. 177). There is little evidence that the ectoderm in general has any capacity to form neural tissue in the absence of an inducing stimulus from the mesoderm, but there is a possibility that the region which develops into the forebrain may be able to do so even when isolated from mesoderm (cf. Waddington 19524, p. 109).
Other evidence of regionality within the organiser emerges clearly from the results of intra-blastodermal grafts. As in the Amphibia, there is a tendency for grafts of the anterior part of the streak to induce heads, 188 PRINCIPLES OF EMBRYOLOGY
whereas more posterior parts do not do so unless they are near the head end of the host. This latter point indicates again an interaction between the host and graft, and in the chick this is sometimes very obvious. When a host embryo and an induced one lie closely side by side, they often ‘fit’ exactly, with each of the organs (head, ears, foregut, heart, etc.) at the same level in each axis, and with the somites exactly lined up (Fig. 10.7). One can find a complete series between entirely separate axes, lying some distance apart, through cases where they are closer and show some degree of fitting, to instances where they have so completely united as to form an almost unitary embryo, whose double origin may be difficult to recognise. However, the experiment described above, in which two epiblasts were placed face to face, shows conclusively that induction is not necessarily dependent on a tendency for a part of the organiser to expand itself into a complete organ or embryo; in that experiment, both streaks were quite complete as regards ectoderm and mesoderm, lacking only their endoderm, whereas what they induced was not the missing endoderm, but was the ectodermal neural system (and probably some mesoderm) which they already possessed.
3. Evocation and individuation
Facts such as these show that one must take account of two aspects of induction, which will have to be explained by two somewhat separate physiological mechanisms because they can be caused to occur independently of one another. The first of these aspects is the mete calling forth of some sort of an induced differentiation—a process which was originally called ‘induction-as-such’ and later ‘evocation’ (Necdham, Waddington and Needham 1934). This is independent of any tendency towards the formation of a complete organic unit, and is, for instance, well exemplified in the appearance of the secondary embryos in the two-epiblast experiment.
The second aspect is the formation of an organised structural entity, which may bea whole embryo, ora part of itsuch asa single organ; for processes of this kind the name ‘individuation’ was suggested (Waddington and Schmidt 1933). The distinction between these two types of process is quite fundamental for any attempt to formulate theories of development which penetrate deeper than the special embryological level to the underlying biochemical or genetical fundamentals. It is important to realise that the characteristic of evocation is not that the response to it is the production of a small or indefinite rudiment (as suggested by Holtfreter 1951) or one which has no definite polarity or structure (cf. Needham 1942, p. 126). On the contrary an evocation may sometimes cause THE EPIGENETICS OF THE EMBRYONIC AXIS 189
the appearance of a well-organised embryo or part of an embryo; but if it does, this embryo must have organised or individuated itself, and its structure will have no connection with any corresponding structure in the evocating material (Waddington 1933b). Evocation is an essentially unitary process, in which one single stimulus calls forth some response; whether the response is simple or complex, organised or disorganised, is another matter. Individuation on the other hand is the process by which a structurally organised entity is built up, and is essentially complex, to a degree which corresponds with the number of elements involved in the organisation. It may occur within a piece of isolated tissue (Fig. 10.9C) or a graft which fails to induce (perhaps because its surroundings are too old); or within the rudiment evocated by a graft which does not take part in the individuation (e.g. a dead graft, Fig. 10.98); or within the graft
Phenomena of individuation. A, gastrula ectoderm, isolated in an evocating solution, has in part differentiated into chaotic neural vesicles, probably representing parts of the brain, which have induced placodes in the epidermis. B, a fragment of adult liver, grafted into an isolated region of gastrula ectoderm, may induce a structure which self-individuates into a wellformed axis. C, an isolated fragment of somite mesoderm from the early gastrula tends to develop into a relatively well-organised axis, with central notochord, accompanied by a neural tube which may swell into a brain-like vesicle at one end; there are muscle cells on each side of the axis, and cephalic neural crest cells at the ‘anterior’. (From Holtfreter, 1951).
and the induced tissues together (Fig. 10.1, p. 175). Holtfreter gives a clear account of these facts; his criticism (1951) of the concepts of evocation and individuation seems to be mainly about the words to be used rather than about the phenomena themselves. 190 PRINCIPLES OF EMBRYOLOGY
Whereas evocation may be, and probably is, a straightforward biochemical process, individuation must always involve a biophysical element, since the organisation of an embryonic rudiment is a matter of geometry as much as of the chemical or histological nature of the tissues. Individuation must also usually involve a number of different biochemical interactions, by which the various tissues comprising the organ are brought into being. For instance, in an induction such as that shown in Fig. 10.1 the combined mass of the graft and the induced tissues have developed into neural plate, notochord, somites and nephric mesoderm. The induced neural tissue is immediately in contact with the graft neural tissue, and the same js true of the induced and graft somitic mesoderm, etc. It seems fairly clear that each tissue developing in the graft must have evocated the formation of tissue similar to itself, Mangold (1932) spoke of such phenomena as ‘assimilative induction’; more recently some authors (e.g. Medawar 1947) have named them ‘infective transformations’, and drawn a parallel with the processes of virus infection from cell to cell. We shall discuss later (p. 401) the grounds which exist for such a suggestion.
Individuation certainly also involves other kinds of biochemical induction besides assimilative evocations. For instance, a well-individuated embryonic axis may be induced by mesoderm which itself forms no neural tissue; indeed this is what happens in normal development. Within the mesoderm itself, inductive phenomena, not of an assimilative kind, can be shown to be involved in its individuation. For instance, presumptive lateral mesoderm, if isolated, develops only into mesenchyme and not into somites or nephros, but if some presumptive notochord is put together with it, the lateral mesoderm is caused to differentiate into one or other or both of these tissues (Yamada 1940). It is as though the notochord were at a high point in a gradient of some kind, the lateral mesoderm at a low one, and in combinations some influence diffuses from the notochord and raises part of the lateral mesoderm to the intermediate level corresponding to somites or nephros (Fig. 10.10).
A considerable amount of study has been devoted to the modification which can be made to the mesoderm gradient field by chemical agents. Lehmann (Review: 1945) has shown that lithium ions acting on the gastrula tend to suppress the development of the notochord; the presumptive chorda cells differentiate into a somitic mesoderm instead of into their usual fate; a similar result can be produced by Trypan Blue (Waddington and Perry, 1955). Ranzi (195 1) found that thiocyanate has the opposite effect of causing a hypertrophy of the chorda. These facts are exactly parallel to those which have been discovered in echinoderm development: THE EPIGENETICS OF THE EMBRYONIC AXIS IOI
it is as though the mesoderm of the amphibian gastrula has the same kind of epigenetic organisation as the whole newly fertilised egg of the seaurchin, the chorda corresponding to the high point of the vegetative gradient and the most ventral mesoderm to the animal pole. Dalcq and Pasteels (1937, 1938, Dalcq 1941) have particularly emphasised this gradient system within the mesoderm, and suggest that it is derived from the two components which were active at the time of formation of the grey crescent, namely, a general gradient in yolk content extending
Differentiation of various regions of the flank mesoderm of the neurula. Column 1 illustrates their presumptive fate; column 2 what they produce when isolated; column 3 what they develop into when combined with a fragment of presumptive notochord. The tissues represented are blood cells, nephric tubules, muscle and notochord. (From Yamada 1940).
through the whole egg from the animal pole to the point of highest concentration at the vegetative pole, and a cortical gradient of unknown nature, located in the outermost layer of cytoplasm and with its highest point at the position where the dorsal lip first appears. The Belgian authors show that one can derive a formal explanation of many phenomena of early development from the interaction of these two postulated gradients; but the concept rather lacks precision when applied to these comparatively late stages of development (see the criticism of Rotmann 1943). The individuation of an organ or of a whole embryonic axis must be highly complex process in which the various parts of the mass of tissue interact with one another in several different types of induction process. Each individual biochemical interaction can, perhaps, be regarded as an 192 PRINCIPLES OF EMBRYOLOGY
evocation, somewhat similar to that by which mesoderm calls forth the production of neural tissue. But the whole complex of such interactions, together with the geometrical aspects of the process, clearly form an organised system which results in the development of an organ with a recognisable structure. For this reason, individuation can be considered as a typical example of a ‘field’ phenomenon.
Nearly all developing masses of tissue exhibit some degree of individuation, which may be a self-individuation (i.e. arising autonomously within the mass) or be partly or wholly imposed on it by an inducer. Individuation is least in evidence in fragments of presumptive ectoderm isolated in salt solution; they form quite disordered epidermal tissues. If they produce neural tissue as a result of the action of a structureless evocator (such as a chemical in solution or a fragment of dead tissue), this may also be almost completely without definite form or arrangement, although even in the most disordered cases there is usually a tendency for the neural cells to arrange themselves into tubules and cysts (Fig. 10.94). Self-individuation may, however, go very much further in such cases, so that definitely recognisable parts of the neural system are formed (c.g. brain, trunk neural tube, etc.); and when the individuation of the earlier stages is fairly well achieved, that of later organs such as the cye, ear, nasal placodes, etc. is often very much better. Isolated fragments of gastrula mesoderm seem always to possess a considerable power of self-individuation, and develop into tissue complexes containing notochord, somites, pronephros etc. with some fairly definite arrangement. It seems likely that the greater tendency to self-individuation in the mesoderm depends on the fact that it develops into several different types of tissue, which can mutually influence one another, whereas the ectoderm tends to form more homogeneous masses. It is noteworthy that the individuation of isolated pieces of mesoderm is better the larger the mass involved, which again suggests that the process depends on interactions between the different parts.
The. phenomenon to which the name self-individuation has been applied here has been particularly emphasised by Lehmann (1945). He suggests that when we are dealing with a small lump of tissue which is starting on a course of development, for instance a fragment of presumptive mesoderm or a region of ectoderm which has responded to an inductor, we should always regard this not as a mere conglomeration of cells, but as a ‘blastema’; and this name, which is the Greek word for a bud, is intended to imply a degree of organisation and an interplay of reciprocal influences between its parts.
Rose (19524) has recently suggested that the appearance of different THE EPIGENETICS OF THE EMBRYONIC AXIS 193
tissues within a self-individuating region depends primarily on the production of inhibiting agents. He supposes that one region will develop fastest, and will differentiate into some specific tissue. He suggests that while doing so, it produces some substance which can diffuse into the surroundings. This substance is supposed, in the first place, to bring the original differentiation process to an end when a high enough concentration is reached, and in the second to impede the tendency of the neighbouring, more slowly developing, tissue to differentiate in the same direction, and thus to swing it over into some other course. The hypothesis makes a pretty and coherent intellectual scheme, but in this simple form suffers from the grave defect of neglecting the fact that all the evidence suggests that embryonic tissues tend to induce the differentiation of their like, rather than to suppress it. Thus, although Rose claims that extracts of adult frog brain will suppress the formation of neural tissue in the embryo, it is more relevant to normal development that young neural tissue induces further similar neural tissue when placed in contact with gastrula ectoderm (so-called ‘homoiogenetic induction’). It is in fact more plausible to suggest that differentiation is usually an ‘autocatalytic process’, the substance produced by one type of differentiation tending to encourage rather than to inhibit the same type of development. The results which Rose deduces from his postulated set of self-limiting reactions would also follow equally from a system of self-reinforcing reactions combined with competitive interactions (see p. 407). Inhibiting substances of the kind postulated by Rose, may however play a part in regulating the growth of the already differentiated tissues of the young adult.
Rose conceives of the inhibiting substances which he postulates as having an immunological specificity, and operating somewhat in the manner of antibodies. It seems rather probable that developing tissues do influence one another (and themselves) by the agency of substances of an immunological character. The possibility has been discussed extensively by Tyler (1947) and Weiss (1947); the latter author has some evidence that adult organs may differentially stimulate the growth of homologous embryonic ones—just the opposite of what Rose suggests. An adequate body of facts in this field is, however, still to seek. But there are indications that a search for them may be rewarding. One may mention the observation of Ebert (1954) recorded on p. 215.
4. The physiology of organiser action a. Natural and unnatural evocators
This analysis of organiser action into two component parts was soon exemplified in quite another way. During the summer of 1932 both the 194 PRINCIPLES OF EMBRYOLOGY
German workers on Amphibia and the British on the chick were successful in obtaining inductions by means of grafts which had been killed (Bautzmann, Holtfreter, Spemann and Mangold 1932, Waddington 1933b). Now it is fairly obvious that whatever a dead graft may be able to induce, it can hardly produce sonicthing which could be regarded as tending to complement the graft and convert it into a complete organic unit, since no dead piece of tissue can possibly form part of a developing embryonic structure. One could therefore consider the possibility that a dead graft might be able to induce something, and even that different regions of a dead organiser might tend to induce different parts of the embryonic axis, but they could certainly not exhibit the whole complexity of the inductive behaviour which is shown, for instance, in the amalgamation of the grafted organiser and what it induces into a complete embryonic axis. In other words, a dead graft might evocate, but it could not individuate. In fact, from the investigation of the capacities of dead organisers, one might hope to arrive at a much more profound analysis of the induction process. Can we perhaps separate evocation again into ‘evocation of some generalised sort of neural tissue’ and ‘evocation of a definite region of the nervous system’: Or is the transmission of regional character always bound up with the completion of a part-structure into an organic whole, and thus necessarily an aspect of individuation? As a matter of fact, we are still not completely sure of the answer (p. 460). The first important advance beyond the bare fact of evocation by the dead organiser was made by Holtfreter (19344, 6). He showed that although, when a graft is made from a living egg, only the presumptive axial mesoderm can induce, the properties of the dead material are rather different; the whole presumptive ectoderm and mesoderm, after killing by heat or organic solvents, will call forth the differentiation of new neural tissue. Moreover, many adult tissues, such as liver or kidney, of the most diverse species ranging through the whole animal kingdom, acted as evocators, particularly when killed before being inserted into the blastocoel of a host egg. This appeared at first sight greatly to facilitate the attempts which several groups of workers were making to extract and identify the evocator substance; instead of starting with dead organiser material, which can only be obtained by dissection of the small gastrula, one could start with large masses of liver and test the activity of various fractions. But the different groups of investigators came to quite different conclusions as to which fractions were the most active. Spemann and his collaborators at first identified the evocator with glycogen; Fischer argued that evocation could be brought about by the stimulus of various acids, among which he mentioned the nucleic acids; Necdham and Waddington THE EPIGENETICS OF THE EMBRYONIC AXIS 195
traced the activity to the fraction of the extract which contained the sterollike substances, and Waddington demonstrated a high degree of activity in certain synthetic substances of the same nature (Fig. 10.11); while Barth suggested that the evocator substance was cephalin (Reviews: Needham 1942, Waddington 19404, Brachet 1944). Obviously not all of these conclusions could be true; and although some of the claims were mistakes based on the presence of impurities, the situation appeared to be one of complete confusion. It only began to clear up when it was shown that
A B FIGURE I0.11
Diagrammatic section showing a neural tube (Ind) induced by a graft (Gr) of gastrula ectoderm which had been cultivated for two days in methylene blue. The graft has also formed some neural tissue. B, A neural tube induced by an implant (Impl) of coagulated albumen containing oestrone. a Waddington, Needham and Brachet 1936 and Waddington 19386).
evocation could be produced by chemicals which quite certainly are not the evocator which occurs in the naturally developing embryo.
The idea of looking for such non-natural evocators arose from another line of thought. In 1927 Spemann and Geinitz had shown that if a piece of ectoderm from the early gastrula is grafted into the region of the organiser and left there for some time, it becomes, as they put it, infected with the ability to induce. Again, various authors have shown that if small fragments of ectoderm are isolated in a situation in which they are bathed by the body fluids (c.g. in the abdominal cavity of a tadpole) they
frequently develop into mesodermal tissues such as are normally derived 196 PRINCIPLES OF EMBRYOLOGY
from the organiser regions (Bautzmann 1929). These facts strongly suggested that the whole of the ectoderm contains all the factors necessary to develop into mesoderm and to induce, and that it is some process occurring at the blastopore region which activates these factors and enables them to take effect.
The well-known axial gradient theory of Child (p. 314) would suggest that the activating process might be something to do with the respiratory metabolism. Waddington, Needham and Brachet (1936) therefore tested the effect on pieces of ectoderm of treatment with dyes known to stimulate respiratory processes. The dye used was methylene blue. It was found, as had been surmised, that if small pieces of ectoderm were isolated in a salt solution containing this substance they sometimes developed into neural tissue, and, if implanted into a young gastrula, were able to induce a neuralisation of the competent ectoderm against which they lay (Fig. 10.11). The dye was, then, acting as an evocator. It would however be ridiculous to suppose that the normal amphibian egg contains methylene blue. It was therefore proved that evocation can be performed by substances other than the substance, whatever it is, which produces that reaction in normal development. This finding put the whole investigation of the nature of inducing action on to a new basis, since, if any substance is grafted into a gastrula and is found to cause neuralisation, that fact cannot be taken as evidence that the substance is the same as, or even necessarily nearly related to, the natural evocator.
There are several mechanisms by which such unnatural evocators might be supposed to operate. In the first place, we have just seen that Holtfreter (1934a) had shown that if non-inducive tissue of the gastrula is killed it thereby acquires the power of induction. There is therefore the possibility that if, in a piece of ectoderm, a certain number of cells were killed they might release sufficient evocating substance to induce the remainder to develop into neural tissue. There is little doubt that processes of this kind can occur. For instance Okada (1938) has brought about inductions by mechanical irritants such as silicious earth and Holtfreter (1945) has done the same thing by killing a certain number of cells with a glass needle. It seems certain however that this is not the only way in which unnatural evocators act. There is no sign of excessive mortality of the cells at the concentration of methylene blue utilised by Waddington, Needham and Brachet; and this has also been pointed out by Pasteels (1951) who confirmed the activity of this substance. Moreover Waddington (1940a) found that the very actively evocating steroid substances tend to stimulate the rate of growth in ectoderm submitted to them rather than to operate as depressants or cytolytic agents. Holtfreter (cf. 1945) held THE EPIGENETICS OF THE EMBRYONIC AXIS 197
for some time to the view that the unnatural evocators acted mainly through the cytolytic mechanism, but he eventually (1948b) found himself driven to speak of a ‘sub-lethal cytolysis’, a somewhat question-begging term which, in effect, admits that the unnatural evocators alter the metabolism of the cells on which they act without actually leading to cell death.
One may take it then that the active substances cause some change in the cell metabolism in the competent ectoderm. We have therefore to conclude that all the factors necessary for development into nervous tissue (and also into organiser derivatives such as chorda, somites, etc.) are already present in the gastrula ectoderm, but require activation before they can be effective.
b. The specificity of the evocator
A certain amount of discussion has gone on in the literature as to whether the unnatural evocators can be considered as ‘specific’ or ‘unspecific’ stimuli. It is rarely that very definite meanings have been attached to these two terms. Perhaps the situation should be envisaged as follows. Let us suppose that, in normal development, a substance, a, diffuses from the archenteron roof into the competent ectoderm and sets going a process, b, which in turn gives rise to process c and d and so on, until neural tissue is fully differentiated. The hypothesis originally put forward by Waddington, Needham and Brachet, and still supported on the whole by Waddington (1940a), Needham (1942), was that substance a already exists within the ectoderm but inactivated in some way, perhaps by being combined with some other substance, «, to form a complex ax. Then the abnormal evocator was envisaged as causing the breakdown of ax and the liberation of the active a. According to this scheme the sequence of processes b, c, d, etc. can only be set in motion by one specific substance, namely a. Alternatively we might suppose that the various unnatural evocators can act immediately on process b, setting it in motion and thus leading to c, d and so on. This would be called an unspecific stimulus because b is supposed to react, not only to a, but to all the other possible unnatural evocators. It must not be forgotten however that even in the first case, although b requires a specific stimulus to set it off, the inactive complex ax is supposed to react unspecifically to any of the abnormal evocating substances. Thus a critical point as regards these two alternatives is whether, when abnormal evocators act on competent ectoderm, a substance appears which is the same as that which normally diffuses from the mesoderm into the ectoderm in normal development. Since, as we shall see, this substance cannot yet be identified, the question 198 PRINCIPLES OF EMBRYOLOGY
is at present unanswerable. The problem of whether the evocator stimulus is specific or not in this sense is therefore, although an interesting one, not profitable to discuss further at the present moment (Fig. 10.12).
There is however rather a different sense in which the terms specific and unspecific can be used. If the evocating stimulus is wholly unspecific, then when used on one and the same type of tissue it can only produce one result. Now this is not the case. We know that in normal development ectoderm differentiates into different parts of the nervous system (e.g.
The activation of the evocator. At the blastopore, something occurs which
converts the inactive tissue into an active evocator (change of (E) to E); this
can then act on the competent tissue C to produce neural differentiation N.
If a foreign substance S is placed in the blastocoel, and produces an induc tion, it might do so either (i) by acting on C (direct evocation), or (ii) by acting on (E) and converting it into E (indirect evocation).
brain, spinal cord, etc.). Further, when it is acted upon in a particular way by the living organiser (c.g. by being grafted into the centre of presumptive mesoderm) presumptive gastrula ectoderm can be caused to develop into notochord, somites, etc. Quite a number of evocators or evocating conditions have now been investigated and it has become clear that they do not all result in the same one out of this gamut of possibilities; they are therefore not unspecific in this sense.
The methylene blue inductions did not live long enough for their detailed characteristics to become clear. Some years later, however, THE EPIGENETICS OF THE EMBRYONIC AXIS 199
Barth (1941) showed that the gastrula ectoderm of the axolotl (A. punctatum) developed sometimes into neural tissue when isolated in, as he thought, completely neutral salt solutions. This was a startling claim because until that time it had always been held that ectoderm could only become neural if definitely induced to do so, and Barth’s result seemed to be putting this in doubt. The matter was reinvestigated by Holtfreter (1945) who found that the truth of the matter is that A. punctatum ectoderm is particularly sensitive to abnormal external conditions and reacted by neuralisation to salt solutions which have no particular effect on the ectoderm of other amphibian species. If however the salt solution is made to depart considerably from the optimum (by a considerable raising or lowering of pH or by lack of calcium), it can evocate neuralisation even in the more resistant ectoderm of other species. Now in such experiments the evocated neural tissue may develop by self-individuation into a fairly well-defined nervous organ. This organ always belongs to the anterior end of the brain (the forebrain or archencephalon). In considerable contrast to this is the result of another type of unnatural evocating condition. Pasteels (summarised: 1953) has shown that fairly mild centrifugation of early gastrula ectoderm will often cause it to develop into neural tissue and also into notochord, somites and other mesodermal derivatives. The ease with which the action is produced varies in different amphibian species. The point to note is that once again definite organs may be produced and, in this case, they never belong to the archencephalon but always to the posterior end of the brain (deuterencephalon) or spinal column. Finally, Yamada (1950) has found that gastrula ectoderm may be caused to develop into mesoderm by treatment with ammonia.
These differences in the results of evocation make rather unplausible the suggestion which has sometimes been put forward (e.g. by Barth) that the evocator reaction is like that of the artificial parthenogenesis. Recent results have only added confirmation to the conclusion reached by Waddington (1940a) that, if the competence of the gastrula ectoderm is set on so fine a hair-trigger that any of a number of stimuli are sufficient to touch it off, we have to admit that the ectoderm can, unlike the egg, shoot in more than one direction. We must in fact be dealing with an orderly system of alternative processes in which the end-result is related in a rather direct way to the nature of the initiating cause. If this were not so, we could expect to get very little profit from an analysis of the evocators, and would have to confine our attention solely to what we can discover as to the processes going on in a reacting ectoderm. As it is we can find important clues to further understanding not only in the ectoderm
oO 200 PRINCIPLES OF EMBRYOLOGY
itsclf but also in the nature of the evocators and in the conditions which convert gastrula ectoderm into organiser.
c. The metabolism of the organiser
It will be convenient next to discuss the latter problem (Reviews: Brachet 1944, Needham 1942, Boell 1948). Child’s theory of axial gradients would suggest that the blastopore region, which is undoubtedly of extreme biological activity, should have a higher rate of respiratory metabolism than the rest of the gastrula; and we have seen that methylene blue, a well-known stimulant to respiratory processes, can cause the release of evocating power in presumptive ectoderm. There are, obviously, considerable technical difficulties in measuring directly the respiration of pieces of tissue as small as the blastopore region, and the first attempts to compare its activity with that of other parts of the embryo led to rather contradictory results. There is no doubt that the consumption of oxygen rises fairly rapidly in the dorsal region of the neurula, when the tissues of the embryonic axis are differentiating. If one wants to assess the metabolism of the organisation centre at the time it exerts its main inductive effect, it is necessary to have an instrument which is sensitive enough to give an accurate reading of the oxygen uptake within the short period of gastrulation. It was not until Needham adapted the Cartesian diver technique of Linderstrém-Lang that this requirement was fully met.
Using this instrument Needham and his co-workers (see Boell, Needham and others, 1939) found that in most series of experiments there was no appreciable difference between the rate of oxygen uptake by the blastopore region and by a piece of tissue from a similar position on the ventral side of the egg. This confirmed the conclusion of earlier work with a lesssensitive instrument by Waddington, Needham and Brachet (1936), but there were other experiments, by Brachet (1936), Brachet and Shapiro (1937), Fischer and Hartwig (1938) which seemed to show a higher activity in the blastopore region. The situation was cleared up by Boell (1942) and Barth (1942), who measured the respiratory rate of a series of isolated fragments from all different parts of the gastrula. They found that there is indeed a gradient in respiration. Its high point, however, is not at the blastopore but at the animal pole, and it falls off from there to reach its lowest in the yolky endoderm. The actual figures given by Boell are: Q’o, (= myl. O2 per pg nitrogen per hour) 4-9 for presumptive neural plate near animal pole, 2-1 for dorsal lip, 2:8 for posterior presumptive ectoderm, 1-3 for endoderm. There is, of course, more yolk in the endoderm cells than in ectoderm, and since this is a relatively inert material THE EPIGENETICS OF THE EMBRYONIC AXIS 201
which would not be expected to consume oxygen, a better comparison would be obtained on a basis of yolk-free cytoplasm rather than of the nitrogen content of the whole cell. An approximation to this can be reached by crushing and centrifuging the various regions of the gastrula and estimating the percentage of volume occupied by yolk. Applying this correction, the Q’o, of the active cytoplasm for the four regions is 7:3, 4-8, 5:2 and 3-8 (Boell 1948) (Figs. 10.13, 10.14). Boell suggested that one should probably also make a further correction for non-yolky but non-respiring cytoplasm; and if this is done, the gradients vanish. Sze
1 1 L | 2 3 4 5
On the left, a section of an axolotl gastrula, divided into regions, whose relative respiratory rates in four different experiments are shown at the right. (From Boell 1948.)
(19534) also finds that in the frog egg, although there are animal-vegetative and dorso-ventral gradients of respiratory activity reckoned on a dry weight basis, all regions respire at the same rate when compared in terms of their content of extractable (=active:) protoplasm. Flickinger (1954) finds that the rates of incorporation of radioactive CO, into the different regions are related in a similar way. It seems then that there is nothing special about the rate of oxygen uptake of the blastopore region; it falls simply into its place in a gradient between the animal and vegetative poles.
The rate of oxygen uptake is, however, by no means the only factor involved in respiratory metabolism. Brachet (1936) found that there is a higher output of CO, from the blastopore region than from comparable 202 PRINCIPLES OF EMBRYOLOGY
ventral regions. Boell and Needham (1939) used the Cartesian diver to obtain more accurate measurements of the respiratory quotient (oxygen uptake divided by CO, output). They found that in the blastula roof the respiratory quotient is about 0-75. By the mid-gastrula, this has risen to 1 in the blastopore region, but is still only about 0-8 on the ventral side, where it rises much more slowly and does not surpass about 0-9 by the end of gastrulation.
A respiratory quotient of unity is often taken to indicate that the respiratory metabolism is involving the breakdown of carbohydrate
}- HQUIOY p10 SA ~ SOLID PERCENTAGE YOLK 80 60 40 20 0 +7— SOLUTION- 5.0F ; k ee SALTS, PROTEIN ETC, 4.0 PRECIPITATED |_-- PROTEIN rte |. —- PIGMENT I 3.0F 0 +4 —HYALINE LAYER ap, Aor Pp — - PIGMENTI ae a YO LEK Lob ° 1 1 i l } 20 40 60 80 100 ‘ACTIVE’ MATERIAL FIGURE 10.14
On the left, a diagram of the stratification of gastrula tissues following high
speed centrifugation. On the right, the respiratory rates of the various
regions of the gastrula plotted against their content of ‘active’ material (i.e. everything except yolk). (From Boell 1948.)
rather than of fat or protein. There is independent evidence that this is in fact the case in the amphibian organiser region. Woerdemann (1933) claimed, on the evidence of histochemical investigations, that glycogen disappeared from the presumptive mesoderm cells as they invaginate through the blastopore. Although Pasteels disputed the validity of his methods, Heatley and Lindahl (1937) demonstrated by microchemical analysis that there is a rapid fall in the glycogen content of the invaginating cells, although it does not disappear entirely.
The Cartesian diver studies also showed that the rate of anaerobic glycolysis is about three times as high in the blastopore region as it is on THE EPIGENETICS OF THE EMBRYONIC AXIS 203
the ventral side (measured in terms of nitrogen content of the whole cell). There is, however, very little glycolysis in any part of the embryo when oxygen is available.
There seems good evidence, therefore, that the organiser region is characterised by a particularly active breakdown of glycogen, although it does not absorb more oxygen than other comparable parts of the embryo. It is tempting to suppose that this carbohydrate metabolism may be connected with the release of the evocator within the invaginating mesoderm; but that conclusion is not the only one which might be put forward. It is, perhaps, even more probable that the breakdown of glycogen provides in the main the energy which must be utilised in the performance of the movements of invagination (cf. Jaeger 1945). The direct oxidation of glycogen is not essential for gastrulation, since many species, particularly of toads, can gastrulate under anaerobic conditions, or in concentrations of cyanide which inhibit 90 per cent of the normal oxygen uptake; some other species (c.g. the frog) are more sensitive and unable to gastrulate under such conditions. (Cleavage is always relatively independent of oxygen in the amphibia). However, Brachet (cf. 1944) found that iodo-acetate, which inhibits the breakdown of glycogen both aerobically and anaerobically, brings gastrulation to a standstill without impairing the inductive power of the organiser. Although the arrest of movement does not occur till the yolk-plug stage, this evidence rather supports the suggestion that the glycogen is being used to provide energy for invagination rather than in direct connection with the evocator.
Barth (sce Barth and Barth 1951) is studying the mechanism by which the embryo utilises the energy derived from glycogen, and also that made available when the yolk is digested at a later stage. He finds that highenergy phosphate bonds, such as those in adenosine triphosphate, are involved, and suggests that the chemical system has some similarity with that characteristic of muscle (see also Dainty et al. 1944 and Fujii et al. 1951).
At the beginning of gastrulation, certain other alterations take place in the metabolism of the egg, and again certain of them appear to go fastest in the blastospore region. A fact which emerges from cytological observation is that in the gastrula the nuclei become suddenly smaller and stain more deeply with DNA dyes (such as the Feulgen reagent) while nucleoli containing RNA make their appearance. These changes presumably indicate an increased synthesis of nuclear RNA and possibly of DNA too (Brachet 19524). They occur more or less simultaneously throughout the animal cells, but more slowly in the endoderm, where the nuclei remain large. Sirlin and Waddington (1954) found, in autoradiographs of 204 PRINCIPLES OF EMBRYOLOGY
embryos treated with radioactive amino-acids, that these are incorporated more rapidly into the nuclei than into the cytoplasm at the early gastrula stage, and that this incorporation, which is probably a sign of synthetic processes, begins first in the dorsal lip region (Fig. 10.15).
A little later, when gastrulation is under way, there seems to be an increase in the cytoplasmic RNA. This can be demonstrated by measuring
On'the left, three diagrammatic sections through the head process (above) the node, and the streak (below) in a chick embryo treated with radioactive methionine. The shading indicates the relative concentration of the tracer, as judged from autoradiographs. (After Feldman and Waddington 1955.) On the right, an autoradiograph of a section through a newt gastrula cultivated ina solution containing radioactive methionine. Theamino-acidhas been incorporated into the proteins, and probably the nucleic acids, particularly in the nuclei of the blastopore region. (After Sirlin, 1955.)
the passage of radioactive phosphorus P** into the RNA fraction of the egg (Kutsky 1950) as well as by histochemical methods. The latter show that the process is not uniform throughout the gastrula, but that the RNA is at first concentrated in the blastopore region. Histochemical methods which reveal protein containing -SH groups give an almost identical picture (Brachet 1944).
Less is known about the metabolism of different regions of the chick blastoderm at the time when the primitive streak is active as an organiser (Reviews: Waddington 1952, Spratt 1952a). Direct measurements of THE EPIGENETICS OF THE EMBRYONIC AXIS 205
oxygen consumption have so far failed to reveal any differences at the various levels along the streak (Phillips 1942), but Jacobson (1938) has shown that there is a rapid disappearance of glycogen during the invagination of the mesoderm, just as there is in Amphibia. Brachet (1944) finds that there is also a concentration of basophilic substances and of -SH containing proteins in the invaginating region, again as in Amphibia; in the chick, in which invagination occurs earlier at the anterior end of the streak, these substances are distributed in a gradient, decreasing towards the posterior. A similar gradient has been found for an indophenol oxidase (probably cytochrome oxidase) by Moog (1943). Rulon (1935) stained blastoderms in oxidised Janus Green, and showed that, under conditions of low oxygen tension, the dye was reduced fastest in the region of the streak; again there was a gradient decreasing from anterior to posterior.
Spratt (1952a, b) has recently studied such reducing systems in more detail, investigating the effects of aerobic as well as anaerobic conditions and varying the nature of the carbohydrate substrates available to the blastoderm. He made the very interesting observation that the node region shows a high rate of activity under a wider range of conditions than does the forebrain; Spratt suggests that we have here a chemical manifestation of the fact that the node is still undetermined and labile, whereas the forebrain has already entered on one particular and limited course of differentiation.
Feldman and Waddington (1955) have studied the incorporation into early chick embryos of radioactive methionine, which presumably gives an indication of the rate of protein synthesis. They found that the incorporation is particularly rapid in the node, and in the thickened ridges on each side of the streak (Fig. 10.15). There seemed to be some loss of the tracer from the newly invaginated mesoderm, which would suggest that a protein is broken down during the invagination process. The metabolism of this amino-acid seems to be of considerable importance, since administration of the unnatural analogue ethionine, which would be expected to interfere with the utilisation of methionine, causes considerable inhibition of development. Herrman (1954) has described rather slighter effects of certain other amino-acid analogues. Very marked inhibition, particularly of the streak and of the somites, is also caused by the purine analogue 8-azoguanine (Waddington, Feldman and Perry 1955). This would be expected to affect nucleic acid metabolism, and probably in this way has an influence on protein synthesis, for which RNA is certainly important. © This short summary is sufficient to make it clear that there are regional differences of metabolism within the avian blastoderm, and that, in 206 PRINCIPLES OF EMBRYOLOGY
general, the most effective inducing region (the node) exhibits the highest metabolic activity. But it is even more difficult for the chick than it is for the amphibian to make any convincing suggestion as to which meta~ bolic peculiarities are mainly responsible for endowing the organiser with its inducing capacity.
5. What occurs during evocation?
The study of the protein metabolism of the embryo is not only perhaps relevant to the activation of the evocator at the blastopore lip, but is even more likely to lead us into the centre of the problem of the nature of the biochemical events concerned in evocation. The differentiation of tissues certainly involves the appearance of tissue-specific proteins and it is probable that an alteration in protein metabolism is one of the most important results of induction. It is, however, extremely difficult to detect small differences in the protein constituents of cells and the analysis of the protein metabolism connected with early differentiation and determination is as yet in its infancy. New techniques will probably have to be elaborated before we can get very far. One of the methods now being explored is paper chromatography. When the proteins of the embryo are hydrolysed and the amino-acids assayed by this method no differences were found either between different stages of development or between different species (Holtfreter, Kozalka and Miller 1950; Eakin 1952). Using fresh unhydrolised tissues, Clayton (1954) has found considerable differences between tissues in fairly young mouse embryos but in these tissue differentiation had already proceeded well beyond the point at which evocator action occurs. Kutsky, Eakin, Berg and Kavanau (1953) have used similar methods in whole amphibian embryos and found a definite sequence of stages as development proceeds. It is probable that further work will on that this method of analysis can give valuable results in the analysis of different regions of the embryo.
Another method which is beginning to be used depends on the incorporation into the proteins of amino-acids labelled with radioactive isotopes. Eakin, Kutsky and Berg (1951) have found that such aminoacids are incorporated more rapidly into dorsal halves than in the ventral halves of the gastrula; and probably this means that proteins are being synthesised more rapidly in the blastopore region. Sirlin and Waddington (1954) have also found that the rate of incorporation varies from tissue to tissue in the early neurula. These two authors, and also Ficq (1954) discovered the interesting fact that the amino-acids are incorporated more rapidly into nucleus than into the cytoplasm in these early stages of development. This may indicate that protein synthesis is actually proTHE EPIGENETICS OF THE EMBRYONIC AXIS 207
ceeding in the nucleus itself, or it may merely mean that amino-acid turnover is more rapid in the nuclear than in the cytoplasmic proteins. As mentioned above, the nuclear uptake begins first in the region of the blastopore, and rapidly spreads into the neural plate. It is probable that in the next few years a great deal of information will become available by this method.
A third method by which a direct approach has recently been made to the problem of protein synthesis as the basis of determination is the use of immunological techniques. Much of the older work in which such methods were used on embryos (Review: Needham 1942) was concerned with the development of species specificity; it is a well-known, though remarkable, fact that tissues of young embryos of quite different species or genera of Amphibia and birds may be grafted together and prove themselves mutually compatible until late stages in development. Even grafts between early embryos of fish and Amphibia or mammals and birds survive for a period which may be quite considerable. Nevertheless recent workers have been successful in demonstrating the existence of organ-specific antigenic substances in very early stages of development, and in using immunological methods to study the changes in such substances as development proceeds. References to most of the recent literature will be found in Woerdeman (1953), Cooper (1950) and Clayton (1953) for the Amphibia and Ebert (1952) for the chick.
Most authors have prepared anti-sera by injecting (into rabbits) minces or extracts of tissues taken from adult animals, and have then tested extracts of embryonic organs to discover whether they react with the anti-serum; if they do, it is concluded that they contain an antigen similar to, or the same as, that in the adult organ. However Clayton (1953) and Flickinger and Nace (1952) prepared their anti-sera by injection of embryos, or parts of them. Clayton made anti-sera against (1) early gastrulae of Triturus alpestris, (2) ectoderm of the early yolk-plug gastrula, (3) archenteron roof of the same stage, and (4) whole tail-buds. When extracts of an embryonic organ were to be tested against these, the whole anti-serum could be used for the test, or it could be first subjected to a process of fractionation by the method of selective absorption; for instance, anti-tail-bud serum can be allowed to react with an extract of gastrula until all the antibodies against antigens present in the gastrula are removed, so that only the antibodies against antigens which arise between the gastrula and tail-bud stages are left. Using this method, some important, though still tentative, conclusions were reached; the reason for hesitation in putting them forward as fully proved is that immunological methods, although extremely sensitive, cannot of course 208 PRINCIPLES OF EMBRYOLOGY
be guaranteed to detect the most minute traces of antigens, and it is possible that substances which seem suddenly to appear during development may have been present in undetectable amounts at earlier stages. Bearing this caution in mind, Clayton’s results were as follows. She was able to detect six different antigenic substances (proteins?) in the blastula to neurula stages. She divided these into (1) a C or common group, which occur in both the ectoderm and mesoderm of the gastrula. Part of this fraction (C) is already present in the blastula, but another part (C*) arises after or during gastrulation, and is presumably synthesised at that time. There are also (2) an E or ectoderm group and (3) an M or mesoderm group, which are confined to the ectoderm or the mesoderm respectively. Again in both these groups there are some components (E and M) which occur in the blastula, and others (Et and M*) which arise during gastrulation. In the blastula, the E and M antigens are thought to exist side by side, only becoming segregated into different tissues as gastrulation proceeds. Similarly, the CC! EE! antigens of the gastrula ectoderm become sorted out between the neural plate and the ectoderm of the neurula. The former probably receives mainly CE! and the latter C'E, but the identifications cannot yet be made with certainty. New antigens also appear during neurulation, e.g. a fraction named Y (Fig. 10.16).
BLASTULA GASTRULA NEURULA TAIL-BUD
Neural plate BC part of C'?
ECTODERM | CICIERE!! Neurula Ectoderm
EAH part of C & E'?
MESODERM CcC' MM!
X Y Z UNALLOCATED consists of C! E' M! not further not further . and perhaps others studied studied FIGURE 10.16
Diagram of the antigenic fractions found in various regions of the newt embryo. (From Clayton 1953.) THE EPIGENETICS OF THE EMBRYONIC AXIS 209
Thus although these studies are as yet in their infancy, they have already yielded considerable evidence that new proteins are being synthesised during gastrulation, and it seems likely that immunological methods will be a very powerful means of investigating the fundamental problem of protein synthesis during development.
It will be noted that Clayton found that the gastrula ectoderm contains certain antigens (C and C!, and E and E') which in the neurula become spatially separated out between the neural plate and epidermis. Her data provide no direct evidence of whether they are already localised in the presumptive areas of the gastrula, but we know that differences in epigenetic behaviour (competence, and capacity for self-differentiation) are only just beginning to arise in gastrulae of this age, and it seems likely that the two groups of antigens, which later become separated, are both present in the same cells in the earlier stage. If this were true, it would be an important fact concerning the mechanism of induction, since we should have to suppose that the antigens characteristic of the neural plate are in some way destroyed or rendered inoperative in the developing epidermis, and vice versa. Some evidence that this may actually be the case can be found in the important studies of Ebert on the chick.
Ebert (1950, 1952) first prepared anti-sera against three adult organs, brain, heart and spleen. The most satisfactory method of testing the embryonic stages for reactivity with these was by adding the anti-sera to agar-albumen clots on which the young blastoderms were allowed to develop. At certain critical concentrations of the anti-sera, rather specific effects were produced on the developing tissues. The sera prepared against mesodermal organs (spleen and heart) tended to prevent the differentiation of mesoderm even at the primitive streak and early somite stages, while having less effect on the nervous system (except in so far as the latter was affected by the suppression of the inducing mesoderm). The two anti-mesoderm sera differed in that the anti-heart serum had a more drastic effect on the heart than the anti-spleen one. The anti-brain serum, at the critical concentrations, had little effect on the mesoderm but suppressed the development of the neural tube. We have then clear evidence that soon after their determination, and during the early phases of differentiation, the different tissues produce different antigenically-active chemical substances, presumably protein in nature.
In later work, Ebert (19536) used sera prepared against the protein (myosin) of adult chick hearts to test various regions of the early blastoderm. He found that there was no reaction by blastoderms younger than the mid-streak stage. By that stage, the invagination of mesoderm has got properly under way, and the mesoderm is probably fully determined 210 PRINCIPLES OF EMBRYOLOGY
to become mesoderm and nothing else; and it is then, at this very early stage, that the immunological techniques show that myosin is already present. In the next stage, of the long primitive streak, Ebert tested not only whole blastoderms but parts of them, as shown in Fig. 10.17. As far as the tests went, myosin was present in all the regions which contain presumptive mesoderm. By this stage, the individuation of the mesoderm
Parts of the chick blastoderm tested immunologically for their content of
cardiac myosin. The intensity of their reaction with test sera is indicated by
the density of dotting. Above, streak and head process stages cut into three
strips. Below, the central strip cut into a middle and two lateral portions. (After data of Ebert 19536.)
is beginning, and different parts of it are acquiring more specific properties (p. 187). By the head process stage, this regionalisation has gone some way; and, further, by this time the presumptive heart mesoderm will have been completely invaginated and moved out laterally to either side of the streak. Ebert finds, in accordance with this, that the myosin reaction has become very weak both in the posterior part of the blastoderm (posterior to the presumptive heart) and in the anterior part of the streak (between the two presumptive heart rudiments), and has disappeared THE EPIGENETICS OF THE EMBRYONIC AXIS 2i1
entirely from the anterior region of the blastoderm. By the next stage (head-fold and early neural groove) it has disappeared also from the anterior part of the streak, and is clearly becoming confined to the actual heart rudiments.
This beautiful series of experiments seems to provide clear evidence that a specific protein can be synthesised throughout the whole of an embryonic region (here the presumptive mesoderm) and then, as regionalisation proceeds within the region, disappear, or at least become undetectable, in all parts except the actual rudiment of the particular organ to which it belongs. It might perhaps be that its disappearance is illusory, and that it merely becomes concealed because it fails, outside its own rudiment, to increase as fast as it does inside it, or as fast as other substances are doing. But the evidence suggests that this is improbable; it seems more likely that the disappearance is a real one. If so, the fact is of capital importance for our understanding of epigenetic processes. It seems quite compatible with the hypothesis (advanced on p. 407) that in development we have to deal with systems of competing processes of such a kind that if one of them gets an initial advantage in a group of cells it will eventually run away with the whole system in that region. Provided that the synthetic processes are reversible, it would be possible for a substance Ato be produced at an early stage, and then, as some other process leading to the formation of B pulled ahead, for the material which had gone into the A channel to be as it were sucked back and taken over by the B process, so that A disappeared again.
Another immunological investigation which has pushed back the detection of specific substances to an early stage, is that of ten Cate and van Doorenmaalen (1950). Using anti-sera against chick and frog adult lenses, they could detect a reaction with the embryonic lens at the stage when it is first becoming detectable anatomically as a vesicle or epidermal thickening.
Clayton (19546) and Clayton and Feldman (1955) have recently introduced a new refinement into the techniques for recognising embryonic proteins by the use of antisera. The antibodies are coupled with either a fluorescent dye or radio-active iodine. When they are then allowed to act on sections of frozen-dried material, the position of the dye or of the radio-active material (which can be made visible by autoradiography) indicates the location of the corresponding antigens.
Besides these direct attacks by new techniques on the problems of protein synthesis in embryos, a considerable amount has been learnt about evocation by older methods. Brachet (1944, 19524, b) has drawn attention to the importance of small granules which can be seen in the cytoplasm 212 PRINCIPLES OF EMBRYOLOGY
in microscopic preparations. These he called microsomes. They can be shown, both by direct chemical methods and by their staining reactions, to be ribonucleo-protein in constitution. They have in general rather low enzymatic activity, but Brachet suggests that they may be the sites at which protein is synthesised. It is worthy of remark that according to a recent report, ribonucleic acid itself shows enzyme activity and is capable of breaking down dipeptides (Binkley 1951); such enzyme actions are frequently reversible and it may be that the nucleotide moiety of the microsomes plays a direct role in coupling together amino-acids to form proteins. The granules certainly increase greatly in number at the beginning of gastrulation, particularly in the organiser region and in the invaginating mesoderm; a little later they appear in large numbers in the developing neural plate. Brachet marshals a large body of evidence, which is in sum quite impressive although unfortunately most of it is somewhat indirect, in support of the hypothesis that these particles play an essential role in induction. For instance, if gastrulae are given a high temperature shock (about 36-37° C., i.e. some 2° C. below the lethal temperature) the gastrulation stops completely and no induction occurs; simultaneously the microsomes lose their nucleic acid. A very similar state of affairs occurs in hybrids between certain species of frogs, in which the disharmony between the cytoplasm and nucleus leads to a blocking of development at the gastrula stage; this evidence shows that the genes must be involved in the metabolism of the microsomes, a point which is of great general importance, as we shall see later (p. 382). In both the heattreated and the hybrid gastrulae the rate of oxygen consumption ceases to rise from the time at which development stops. [Barth and Sze (1951) have shown that the summed oxygen uptake of a piece of organiser and a piece of gastrula ectoderm is higher when they are placed together so that an induction can occur than it is when they are kept separate. But it is not clear whether the extra oxygen is used for the actual evocation itself, or for the neural differentiation which the induced ectoderm undergocs.] It is rather surprising to find that these changes can often be reversed and development be started up again if a fragment is transplanted to a normal host embryo, which must be able to supply some essential substance lacking from the blocked tissues. The nature of the substance is obscure.
Again, Holtfreter (1945, 1948b) showed that when gastrula ectoderm is submitted to abnormal media (producing so-called sub-lethal cytolosis) there is a massive appearance of microsome-like bodies in the cytoplasm. Waddington and Goodhart (1949) made much the same observation when studying the mode of action of the steroid-like evocators. The location THE EPIGENETICS OF THE EMBRYONIC AXIS 213
of these substances in the cell can be revealed by their fluorescence when illuminated with ultra-violet. It was found that when applied to amphibian gastrula cells they become, in the first place, attached to lipochondria, that is, to cytoplasmic granules which contain considerable quantities of lipid as well as protein. In normal development, as Holtfreter (1946) showed, these granules break down at just the time when the microsomes are increasing in number during gastrulation and it seems likely that the microsomes are, in fact, produced by the dissociation of the lipochondria into their lipid and protein parts. Under the action of the steroid evocators the breakdown is accelerated. Finally, Pasteels (1951, 1953) has noticed a similar appearance of basophilic cytoplasmic granules in gas~ trula cells which have been centrifuged and thus caused to undergo spontaneous neuralisation.
All these observations provide considerable support for Brachet’s suggestion that the microsomes are intimately involved in the reaction of the gastrula ectoderm to evocatory stimuli. They are probably in fact the site of the reactions b to c to d, etc. as postulated above (p. 197). This accords well with the evidence (pp. 90, 101) that centrifugable granules are important in determining the characters of the different regions in mosaic eggs, the gradients in echinoderms, ete.
Brachet also suggests that microsome-like bodies not only constitute the system which controls the competence of the reacting ectoderm but are also the natural evocator itself, that is to say the substance we symbolised as ain the scheme above. He brings forward several pieces of evidence in support of this. Firstly, he claimed (1944) that if dead tissues, capable of evocating, are digested with ribonuclease they lose their inducing power at the same time that the microsomes granules are destroyed; but more recent work (Brachet, Kuusi and Gothié 1952) has shown that this is not necessarily the case; it may be the protein, rather than the RNA which is effective. Secondly, he points out that in normal development there is a considerable accumulation of basophilic granular material between the invaginated mesoderm and the overlying ectoderm and although it is difficult to be certain, there is some suggestion that these particles are actually passed from the lower layer of tissue into the upper. Thirdly, Brachet stained living mesoderm with a vital dye, neutral red, which attaches itself mainly to the microsome granules. When this mesoderm is placed in contact with reactive ectoderm the induction takes place and at the same time the red colour is seen to pass into the reacting tissue. This might of course be a mere diffusion of the liberated dye itself. That possibility is rendered improbable by the observation that if a porous membrane, through which the dye molecules can pass, is placed between the 214 PRINCIPLES OF EMBRYOLOGY
mesoderm and the ectoderm, no induction takes place and neither does the colour become transferred from one group of cells to the other. This seems to indicate that the transfer of dye is brought about by the passage of rather large particles which are not able to pass through the membrane, and thus supports the suggestion that particles of the size of microsomes are able to migrate from cell to cell. None of these observations are, however, as clear cut as one would desire to establish such an important conclusion as the intercellular migration of large microsome-like particles.
Niu and Twitty (1953) have recently made the important observation that if pieces of axial mesoderm are cultured in saline solution for some days, and small fragments of gastrula ectoderm then added to the culture, these may become induced to differentiate into neural tissue, even when they do not establish any direct cell-to-cell contact with the original mesodermal explant. The induction must be due to substances given off by the mesoderm into the culture medium. It is possible that these substances are relatively unspecific and act through a relay mechanism (as would, for instance, acids) but it seems more probable that we are really confronted here with a diffusion of the normal evocator itself. Preliminary spectroscopic study shows that the medium, after conditioning by the mesoderm implant, contains substances which may be nucleic acids; but the biochemical analysis is still in a very early stage. It has been mentioned earlier (p. 196) that Bautzmann (1929) had already shown that the body fluids of older larva exert an organiser-like influence on fragments of ectoderm isolated in it.
Some attempts have been made to get further information by the use of radioactive labelling. Waddington (1950b) showed that, if yeast is labelled by being cultivated for some time in solutions containing phosphorus-32, then dried and used as a graft in the gastrula, the radioactive material passes from the implant into the neuralising ectoderm. It is not clear however whether the phosphorus is carried by large complexes containing nucleic acids, or whether it is in the form of small groups such as the phosphate ion when it makes the passage from one tissue to the other. Ficq (1954) has also used organiser grafts labelled by cultivation in radioactive amino-acid solutions. She found that after a few days the radioactivity was no longer solely confined to the graft. It occurred in the evocated neural tissue, but it was also found in the neural system of the host embryo. It is therefore probable that we are dealing not with a straightforward diffusion of the radioactive material out of the graft into the surroundings, but rather with a selective accumulation by the most rapidly metabolising tissues of substances released from the graft itself, which become available throughout the whole embryo. Again it is unTHE EPIGENETICS OF THE EMBRYONIC AXIS airs
clear whether these substances are in the form of individual amino-acids or of larger, more complex particles. There is some evidence that, at later stages, after the blood circulation is established, different organs may give off substances which are complex enough to carry tissue specificity. Thus Ebert (1953) reports that if grafts are made on to the chorio-allantoic membrane of the chick, with fragments of spleen, liver or kidney from adult fowls injected with methionine-S*°, the radioactivity is found later to be specifically accumulated in the embryonic organ corresponding to the graft; that from spleen grafts goes primarily to the embryonic spleen, from kidney to the embryonic kidney, and so on. However, Sirlin and Waddington (1955) were not able to find any evidence of such organspecific transfers of substances in the early stages of the amphibia, before the onset of circulation. They could also not confirm Ficq’s observations of a passage of the tracer from a labelled organiser into the host neural tissue as well as into the induction. They point out that the tracer which gets into the induced tissues is mainly in the nuclei, and therefore gives no evidence for a passage of cytoplasmic microsomes. They suggest, indeed, that the tracer may actually be passing in the form of the free aminoacid, in which case it will yield little information about the mechanisms of induction.
Abercrombie and Causey (1950) have used radio-phosphorus to label regions of the chick primitive streak which were then used as inducing grafts into other blastoderms. They were able to distinguish the graft tissues fairly sharply from those of the host but their technique was not
adequate to decide whether any minor spread of labelled compounds
from the graft had taken place.
Thus the experiments using radioactive labelling have not yet given unequivocal evidence that bodies as large as microsomes pass from the mesoderm into the ectoderm during evocation, but neither do they refute the suggestion.
6. Regionally specific evocation
As was mentioned earlier, Spemann and many later authors have shown that the first invaginated, presumptively anterior, part of the mesoderm has a strong tendency to induce the anterior part of the nervous system, while the presumptively posterior mesoderm tends rather to induce trunk or tail regions. These phenomena present us with two rather different types of problem. On the one hand there is the question of whether the anterior and posterior parts of the organiser owe their specific effects to chemical differences in the evocators which they release. This is important for our present discussion, since if it were true it would be good evidence
P 216 PRINCIPLES OF EMBRYOLOGY
that evocation is dependent on specific rather than unspecific stimuli. But however much we might discover about the chemical differences within the organiser, this would not provide any explanation of the way in which the reacting tissue comes to assume the specific and definite shape of a particular neural organ such as the forebrain, hindbrain or spinal cord, or of how the evocating substances come to be arranged in an orderly pattern within the mesoderm. This is the problem of morphogenesis and individuation. It will be discussed more fully later (p. 455). Here we shall deal only with the simpler chemical problem of the nature of the evocators involved.
There is considerable evidence (see Holtfreter 1951), that competent ectoderm of the gastrula has no particular regional properties and that no part of it shows any special tendency to develop either towards the head or the tail. Although Nieuwkoop (1947) fairly recently queried this, he seems to have had little good reason for doing so, other than the needs of a theoretical scheme he was putting forward. If therefore a process of induction shows a regionally specific character, this is to be attributed to the properties of the inducer rather than of the reacting material.
Early attempts (Lopaschov 1935a, b) to demonstrate the existence of chemically different evocators in different regions of the mesoderm were rather unsuccessful. However, shortly after this Chuang (1938, 1939, 1940) and Toivonen (1940) found characteristic regional differences in the inductions produced by implants of killed tissues from various organs of adult mammals (see also Hama 1944). The subject has since been rather thoroughly investigated by Toivonen (summary 1949, 1950). There is no close anatomical correspondence between the organ from which the implanted tissue is taken and the type of induction it produces. For instance, Chuang found that mouse kidney tended to induce parts of the brain, and Triton liver induced more posterior structures; while according to Toivonen, a guinea-pig kidney is a posterior inductor and guinea-pig liver an inductor of anterior parts. The distribution of the evocator substances in the adult body therefore seems to be rather haphazard.
Toivonen has made some progress towards finding out the chemical properties of the substances concerned. He concludes that there are two main evocators. The property of one is to induce anterior regions of the brain (the so-called archencephalon). This substance is relatively resistant to boiling, is soluble in organic solvents and easily dialysable. The second substance induces parts of the spinal column or the hindbrain (the deuterencephalon). It is very thermolabile, being destroyed by heating to 90° C. and is insoluble in petrol ether. The adult tissues as used in imTHE EPIGENETICS OF THE EMBRYONIC AXIS 217
plants nearly always contain mixtures of these substances, one or other predominating in particular cases; it is possible, for instance, to reveal the presence of small amounts of the posterior-inducing substance in guinea-pig liver tissue when the brain-inducing material is removed by thorough extraction with petrol ether.
The existence of these two different evocators seems rather thoroughly established by Toivonen’s data. There are however several questions about them which still remain open. In the first place, should they really be regarded as specific inductors of particular regions, rather than of particular tissues? It is noteworthy that the archencephalon is the part of the nervous system which, in the normal embryo, is not accompanied by any mesoderm: and when posterior parts of the neural system are induced, some induced mesodermal structures always appear with them. One might in fact suggest (cf. Waddington 1952c) that the archencephalic evocator is a pure neural inductor while the spinal inductor is able to induce mesoderm as well as neural tissue, the anterior or posterior character of the induced organ depending on the proportion between the amounts of induced neural and mesodermal material. Toivonen (1953) has in fact recently found that alcohol-fixed bone marrow is a specifically mesodermal inductor causing the appearance of induced muscles, extremities, etc., unaccompanied by any induced neural tissue.
Another problem is the relation between these evocators from adult tissues and the factors active in the invaginating mesoderm of the gastrula. It has been known since the early work of Holtfreter (1934) that the organiser material of the gastrula, when extracted with boiling water, alcohol, etc., retains its power to induce neural tissue but loses that to induce mesoderm. Barth and Graff (1943) showed that the same is true if the organiser region is freeze-dried. Waddington (1952c) re-examined the effect of slight heat treatment. The abolition of mesoderm-inducing capacity was confirmed and the evidence suggested that the neuralinducing capacity is not in any way regionally differentiated. Large masses of induced neural tissue sometimes form themselves into archencephalic vesicles (i.e. forebrain) ; and thus it seems not unreasonable to suppose that the heat-resistant neural evocator of the gastrula mesoderm is the same as the archencephalic evocator isolated by Toivonen from adult tissues. Lallier (1950) claims that the neural-inducing capacity of the gastrula organiser is abolished by treatment with formalin, but it is not clear whether this is true of Toivonen’s archencephalic evocator. The mesoderm-inducing capacity of the living gastrula organiser also shows properties not unlike those of the spinal inducer postulated by Toivonen. In particular they are both heat labile. We shall see later when discussing 218 PRINCIPLES OF EMBRYOLOGY
the individuation of the induced axis (p. 462) that there is evidence that the mesodermal inductor (or trunk inductor) acts after the archencephalic or neural inductor and probably antagonistically to it. There is not yet any corresponding evidence as to the time of action of the adult evocators, but in general it seems not improbable that Toivonen’s adult evocators are at least very similar to, if not identical with, substances which are active during gastrulation.
Kuusi (1951, 1953) has studied the evocatory powers of various fractions (nuclei, microsomes, plasma, etc.) of the guinea-pig liver and kidney tissue. The results are not very easy to interpret but she comes to the tentative conclusion that the spinal (mesodermal) inducer is probably a protein while the archencephalic (neural) one may be represented by the microsomes.
A point which still requires considerably further study is the exact mode of action of the two classes of evocators. The gastrula ectoderm is normally two-layered, with an outer ‘epidermal’ layer and an inner ‘sensory’ one. In the normal induction of the neural plate, both layers become converted into neural tissue, but when abnormal evocators are used, their effect is sometimes confined to the inner sensory layer. Fujii (1944), comparing inductions produced by the coloured dorsal skin of the adult frog (which tends to evocate neural tissue) with those by the white ventral skin (which tends to induce mesoderm), raised the question of whether the two evocators act differentially on the two components of the ectoderm. Fujii’s inductions were rather feeble, and his material not very convincing, but the problem would seem likely to repay further study.
The fact that it has been necessary to discuss at some length whether there is or is not any degree of specificity in the evocator(s) is sufficient to emphasise the great part played in development by the competence of the reacting tissues. What does this competence consist of, and what can be learnt about its behaviour? (Reviews: Waddington 19404, Holtfretcr 1951).
Competence, as was said above (p. 179), is a state of instability between certain alternatives. The gastrula ectoderm is competent with respect to the broad alternatives of epidermal, neural and mesodermal differentiation. Within each of these main categorics there are certain subdivisions: for the first, true epidermis, lens placode, ear placode, etc.; for the second, the various regions of the brain, the spinal cord, neural crest, etc.; for the third, chorda, somites, nephros, lateral plate, etc. The state of instability THE EPIGENETICS OF THE EMBRYONIC AXIS 219
between these sub-alternatives persists longer than that between the main ones; thus pieces of tissue which have already become neural plate (and thus entered on one of the main alternative paths) may still for a short time be responsive to influences tending to change the region of the neural system which they will form. There is a considerably longer interval between the time at which the non-neural ectoderm is delimited and that when it is decided whether it shall become lens or ear. We have then a succession of competences in time.
The first study devoted to the causal relations, if any, between successive competences was made in the chick (Waddington 19342). In that form, as we have seen (p. 182), the embryonic endoderm induces the formation of the primitive streak; does it also provoke the arising of neural competence in the ectoderm? If so, this competence should be absent in the area opaca, outside the region occupied by embryonic endoderm. It was found, however, that the area opaca reacts to organiser grafts just as well as does the area pellucida. The neural competence therefore seems to arise independently of the endoderm.
A similar investigation was made on the Amphibia (Waddington 1936). Pieces of ectoderm were removed from the gastrula before the organiser had acted on them, and were cultivated in isolation in salt solution until control embryos of the same age had reached the neural plate stage (i.e. had completed the primary organiser action) ; fragments of anterior neural plate were then implanted into them. The first fact that emerged was that the neural competence had been to a large extent, though not completely, lost. There is therefore an autonomous lapse of competence, although this is not as rapid as it would be in a complete embryo in which organiser action had taken place. Secondly, it was found that when the implanted anterior organisers developed into eyes, they were often able to induce the formation of lenses from the isolated ectoderm, in which lens competence must therefore have arisen independently of the action of the primary organiser. In more extensive experiments, Holtfreter (1938)) obtained similar results on the loss of competence, and showed that this is a gradual, not a sudden occurrence; as the ectoderm ages, the magnitude of the neural inductions diminishes, and one obtains more of the ‘weaker’ reactions such as the derivatives of the neural crest. The observation of Pasteels (1953) that centrifugation in the blastula stage often causes the appearance of mesodermal as well as neural tissues, while later only neural, and finally neural crest derivatives appear, is probably also to be explained by the waning of neural competence, but in this case the equivalence of the treatments applied at the different stages is not so certain, since the cells may change in their internal viscosity. 220 PRINCIPLES OF EMBRYOLOGY
There is still very much to learn about the origin and loss of competence, and its quantitative intensity. It is, for instance, difficult to believe that all competences throughout development can arise autonomously, without dependence on previous inductive processes. There were indeed some indications in Waddington’s experiments that the lens competence is not completely autonomous, since it seemed only to appear if the isolated ectoderm remained as a fairly thin sheet, and to be absent from thick _ solid masses of tissue (for discussion: see Waddington 1940a). Another very interesting problem is that implicit in the use of the phrase ‘weaker reactions’ above. Is the decision between the sub-alternatives dependent on the strength (i.e. intensity and/or duration) of stimulus? and can variations of this kind perhaps even tip the scales between the major alternatives of neural tissue and mesoderm? We shall return to this question in discussing individuation of the embryonic axis (Chapter XX).
It must not be overlooked that competence involves the capacity for self-individuation leading to the formation of a well-shaped organ. In the normal development of the neural system, the inducing archenteron roof undoubtedly plays an important part in the individuation of the neural system, but quite well-organised differentiation can occur even when the inducer is certainly structureless and unable to contribute to the result.
Although so little progress has been made with the embryological study of the waxing and waning of competence and the factors which bring it into being, there is another line of approach to which we may turn. Granted that competence is a state of instability in a complex system of reactants, what may we suppose these reactants to be: Now genetics has taught us that the characters which an egg develops are ultimately controlled by the genes contained in its nucleus. The various processes which may or may not proceed, according as the instability is resolved one way or the other, must therefore be gene-controlled; and the reactants which give rise to the competence must be the genes or at least factors dependent on the genes. Evidence of this may be seen within the organiser phenomenon itself. We have mentioned several times that organiser grafts may be active even if made between quite different species. If ectoderm from a species of Urodele with a large egg is grafted so that it comes to lie over the mesoderm of a small egg of another species, it is found that only a small neural plate is formed; there is an adjustment to the size of the inducing organiser out of which the evocator diffuses (Holtfreter 1935). But’ this is almost the only respect in which such an adjustment occurs. As regards nearly all other characters but size, the induced organs have the characteristics of the species to which the competent tissue belongs (for discussion: THE EPIGENETICS OF THE EMBRYONIC AXIS 221
see Baltzer 1950, 1952). This is not so clear for the neural tissue, where there are no very obvious differences between the available species of Amphibia. But as we shall see, there are secondary organisers which act later to induce structures where such differences may be more marked. For instance, the mouth of an early frog embryo has proper teeth, whereas that of a newt does not; and if a newt mouth-organiser acts on frog ectoderm, the result is a frog mouth, complete with tecth (Spemann and Schotte 1932, Rotmann 1935). The inducing stimulus gives the order ‘Mouth’, and the reacting ectoderm carries out the order in accordance with its own book of procedure. The particular substances formed during the differentiation are determined, in the main, by the genetic nature of the competence. Another example of this is illustrated in Fig. 10.18.
It is worth noting that, as might perhaps be expected, Briggs, Green and King (1951) found no sign of any competence in non-nucleated amphibian cells, of the kind mentioned on p. 64.
It will be apparent from this discussion that the investigation of evocation has led us into the very heart of the complex metabolic life of the
A graft of newt ectoderm (pale) was made on to a toad embryo. Where the implant comes into the mouth region, it has developed a balancer, an organ which a newt embryo would possess in this region but which is quite foreign to the toad. Thus the implant has reacted in its own characteristic manner to the stimulus of the region. (After Rotmann 1941.) 222 PRINCIPLES OF EMBRYOLOGY
cell. We are not dealing with a simple interaction between a single stimulating substance and a definite and delineated reactant. On the contrary, a choice as fundamental as that between epidermis and neural tissue involves the whole biochemical system of the cell; the protein synthesis, the ribonucleo-protein microsomes, the respiration and the genes. It is perhaps disappointing that the complexity of the system makes it impossible for us to reach a quick and definite identification of ‘the evocator’. But in compensation we are acquiring a much more profound insight into the whole economy of epigenctic processes. Before we can advance any further, it will be necessary to discuss more fully the body of knowledge which genetics can bring to bear on the problem, which will be done in Part II of this book.
The classical papers on organisers are Spemann 1918, Spemann and Mangold 1924. Spemann’s own summary of his work is in his 1938 book, Chapters 6-14.
For recent accounts of the experimental facts: Needham 1942, pp. 148-205 and 27189: Lehmann 1945, pp. 203-350; Waddington 19524, pp. 51-139. For biochemical aspects, Brachet 1944, Chapters 9 and 10, Boell 1948. Other valuable discussions, Brachet 1952a, b, Holtfreter 19486, 1951, Toivonen 1950.