Waddington1956 1

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Waddington CH. Principles of Embryology (1956) The MacMillan Co., New York

   Principles of Embryology (1956): Part 1 - 1 The Science of Embryology | 2 The Gametes | 3 Fertilisation | 4 Cleavage | 5 The Echinoderms | 6 Spirally Cleaving Eggs | 7 The Ascidians and Amphioxus | 8 The Insects | 9 The Vertebrates: The Amphibia and Birds | 10 The Epigenetics of the Embryonic Axis | 11 Embryo Formation in Other Groups of Vertebrates | 12 Organ Development in Vertebrates | 13 Growth | 14 Regeneration | 15 The Role of Genes in the Epigenetic System | 16 The Activation of Genes by the Cytoplasm | 17 The Synthesis of New Substances | 18 Plasmagenes | 19 The Differentiating System | 20 Individuation - The Formation of Pattern and Shape | References
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The Science of Embryology

1. The place of embryology among the biological sciences

The core of the science of embryology is the study of developmental phenomena in the early stages of the life-history of animals. It is, however, impossible to discover any general and important dividing line between the embryonic and later stages of development, and there is no good reason to exclude from the purview of the subject those processes of development which take place in stages later than the strictly embryonic. It is best, in fact, to understand the word ‘embryology’ as referring to all aspects of animal development, in which case it will include, among the peripheral fields in which it shades off into other sciences, some phenomena which may also be considered as parts of endocrinology or of genetics.

From whatever point of view one regards the biological sciences, the study of development will inevitably be found to take a central position among them. If one attempts to view biology as a whole, there are broadly speaking two main approaches which one can adopt; either one tries to formulate a general system which will exhibit all the major aspects of animal existence in their proper relation to one another; or one searches for a theory of ultimate units which could play the same role for biology as the electrons and similar particles do for physics and chemistry.

From the first, or synthetic, point of view, the most fundamental character of living things is the way in which time is involved in their existence. An animal functions from minute to minute or from hour to hour, in feeding, digesting, respiring, using its muscles, nerves, glands and so on. These processes of physiological functioning may be repeated within periods of time which are short in comparison with the lifetime of an individual animal. But there is an equally important set of processes, of a slower tempo, which require appreciable fractions of the life-history and are repeated only a few times, if at all, during one life-cycle; these constitute development. Still longer-term processes are those of heredity, which can only be realised during the passage of at least a few generations and which form the province of genetics. And finally, no full picture of an animal can be given without taking account of the still slower processes of evolution, which unfold themselves only in the course of many lifetimes. From this point of view, then, embryology takes its place between physiology on the one side and genetics on the other.

As a matter of historical fact, the biological sciences at the two ends of the time-scale—those of physiology in the broad sense on the one hand, and of evolution on the other—have been more thoroughly developed than the two sciences of embryology and genetics which come between them. The volume of information available about physiological phenomena is immense; their relevance to medicine and animal husbandry has given them practical importance, and the relative ease with which they can be envisaged in physico-chemical terms has made them seem intellectually attractive. The study of evolution, which was until recently only slightly less voluminous, derived its impetus from the feeling that Darwin’s work has provided the essential thread which was needed to link all aspects of biology together. Between these two huge masses of biological science, embryology and genetics are rather in the position of the neglected younger sisters in a fairy tale.

At the present time it looks rather as though the fairy tale will have the conventional ending, and the elder sisters find themselves in difficulties from which the younger ones will have to rescue them. This is becoming most apparent in connection with evolutionary studies; their enormous expansion in the past has been mainly by the essentially non-experimental methods of comparative anatomy and taxonomy, and it is already clear that little progress can be made towards an understanding of the causal mechanisms of evolution without the aid of genetics and to a lesser extent of embryology. And even physiology finds itself more and more led to the recognition that structural considerations are of the utmost importance for the functioning of biological systems; and this realisation brings it into close contact with embryology, which of all the biological sciences is most concerned with questions of structure and form.

The central position of embryology is perhaps better appreciated when one regards biology from the other viewpoint, which seeks to discover some category of ultimate units. It is clear that the unit which underlies the phenomena of evolution, and of the short-term heredity which constitutes genetics in the narrow sense, is the Mendelian factor or gene. But any theory based on our present knowledge of genes has perforce a most uncomfortable gap in it at the place where it should explain how they control the characters of the animals in which they are carried. For physiology, the basic unit is the enzyme. We know that the formation of most, if not all, enzymes is controlled by genes; in fact it is not unplausible to suggest that genes are simply a particularly powerful class of enzymes. But here once again we find ourselves confronted with that most lamentable deficiency, our lack of knowledge of exactly what genes do and how they interact with other parts of the cell in doing it. But whatever the immediate operations of genes turn out to be, they most certainly belong to the category of developmental processes and thus belong to the province of embryology. This central problem of fundamental biology at the present time is of course being attacked from many sides, both by physiologists and biochemists and by geneticists; but it is essentially an embryological problem.

It is unlikely that the methods of classical descriptive or experimental embryology will suffice to bring any solution to the problem of the genetic control of development. Neither will the conventional breeding methods of classical genetics, or, in all probability, the normal techniques of biochemistry and physiology. A general textbook of embryology can, however, not be confined to those novel techniques of investigation which, at any given time, seem most likely to lead to major advances in understanding. New methods can usually only be applied to old material; and new ideas do not suddenly emerge full-fashioned, as Aphrodite was born from the chaotic sea; they are built up laboriously on the foundation of previous work. Thus this book will attempt to describe, in the abbreviated and simplified outline which considerations of space impose, the general framework of embryological science within which the attack on the fundamental problems has to be made. Those problems cannot always be in the forefront, but the importance of the various aspects of embryology will be better appreciated if one has a clear realisation of the nature of the goal towards which our expanding knowledge is advancing.

2. An outline of development

Since all animals are in some way related, through the processes of evolution, there are some similarities in their various forms of development. One can, in fact, sketch a broad outline of the early stages of development which applies, roughly at least, to all the animal phyla. This can best be described in terms of a series of stages:

Stage 1. The maturation of the egg. The period during which the egg-cell is formed in the ovary might be thought to come, as it were, before embryology begins, but actually it is of great importance. It is, of course, the time when the meiotic divisions of the nucleus occur and the number of chromosomes is reduced to the haploid set. Further, the egg is pumped full of nutritive materials of various kinds, collectively known as ‘yolk’ (in the broad sense of that word); there are usually special ‘nurse cells’, closely applied to the growing egg in the ovary, which are concerned in supplying these stores of yolk. Finally, and most important of all, it is during this time that the egg-cell acquires its basic structure, which provides the framework for all the elaboration which will occur in later development. This basic structure always involves a polar difference by which one end of the egg becomes different to the opposite end; these are the so-called animal and vegetative poles. There may be also a second difference, distinguishing the dorsal from the ventral side and thus defining a plane of bilateral symmetry; perhaps indeed there is always some trace of such a difference, though it is not always well marked or very stable. Lastly one may mention a difference of another kind, between a cortex which forms the outer surface of the egg and an internal cytoplasm which is usually more fluid. We shall see that all these three elements of structure—the animal-vegetative axis, the dorso-ventral axis and the cortex-cytoplasm system—play very important roles in development.

Stage 2. Fertilisation. This stage involves two important processes; the union of the haploid nucleus carried by the egg with that of the sperm, and the ‘activation’ of the egg, which causes it to begin dividing and thus to pass into the next stage. These two processes are distinct from one another, and we shall see that activation can happen without any union of the nuclei taking place.

Stage 3. Cleavage. The egg-cell becomes divided into smaller and smaller parts by a process of cell division. There are many different patterns in which such cleavage can occur, and it is greatly influenced by the presence of large quantities of yolk.

Stage 4. The blastula. Cell division continues throughout the greater part of the embryonic period, but the stage of cleavage is said to come to an end when the next important developmental event occurs. This event is gastrulation, and the embryo which is just ready to start gastrulating is spoken of as a blastula. In its most typical form the blastula consists simply of a hollow mass of smallish cells; these have been produced from the egg by cell division, and the hollow space in the middle of the mass is formed by the secretion of some fluid material into the centre of the group. When there is a considerable quantity of yolk, the blastula becomes asymmetrical, the cells which contain a high concentration of yolk being larger than the others. In the extreme case, such as in the eggs of birds, the yolky end (the vegetative pole) does not cleave at all, and the blastula becomes reduced to a small flat plate floating. on the upper pole of the yolk; this is known as a blastoderm.

Stage 5. Gastrulation. In a short and extremely critical period of development, the various regions of the blastula become folded and moved around in such a way as to build up an embryo which contains three more or less distinct layers (only the inner and outer layers appear in coelenterates and lower forms). These three fundamental layers are known as (i) the ectoderm, which lies outermost, and will develop into the skin and the neural tissue, (ii) the endoderm, which lies innermost and will form the gut and its appurtenances, and (iii) the mesoderm, which lies between the other two, and will form the muscles, skeleton, etc. The foldings by which these layers are brought into the correct relation with one another are very different in different groups, as they are bound to be since the blastulae from which they start may not have the typical spherical shape, particularly when there is much yolk in the egg. But in spite of differences in the process of gastrulation, the situation to which it leads—one in which there is an outer, an inner and a middle layer—is rather uniform in all groups.

Stage 6. Formation of the basic organs. Soon after gastrulation the fundamental pattern of the embryo begins to appear. In most cases, the organs which arise are ones which will persist throughout the remainder of development, and will form the most essential organs of the adult animal; but in some animals the embryo at first develops into a larva, forming organs which require radical alteration before the adult appears. There are, of course, too many types of adult or larva in the whole animal kingdom for it to be possible to give a single scheme of basic organs which can apply to them all, but it is perhaps worth while to indicate the general pattern of all the various types of vertebrates. Such a scheme is shown in Fig. 1.1. We see that the ectoderm forms, firstly, the skin which covers the whole body, and secondly a thickened plate which folds up to form first a groove and finally a tube which sinks below the surface and differentiates into the central nervous system. (At the boundary between the neural and skin parts of the ectoderm, cells leave the ectodermal sheet and move into the interior of the embryo; this ‘neural crest’, which forms nervous ganglia and other organs is not shown in the Figure.) The sheet of mesoderm becomes split up longitudinally into a series of zones. Under the midline of the embryo is a long rod-like structure, the notochord, which is the first skeletal element to appear. On each side of this the mesoderm is thickened and transversely segmented so that it takes the form of a series of roughly cuboidal blocks, which are known as somites, and which give rise to the main muscles of the trunk as well as the inner layers of the skin. Laterally on each side of the somites there is a zone of mesoderm which will later produce the nephroi or kidneys, and laterally again more mesoderm which is not transversely segmented and which is destined to give rise to the limbs and the more ventral muscles and sub-epidermal skin. Finally, in the most inner recesses of the embryo, the endoderm becomes folded into a tubular structure which is the beginning of the gut or intestine. The formation of these organs always begins earlier in the anterior end of the embryo than in the posterior.


FIGURE 1.1 To illustrate the basic structure of a generalised vertebrate embryo.


After these basic elements in the adult structure have been roughed out, there remains, of course, much to be done in adding the details, but the phenomena differ so much in the various phyla that there is no point in trying to describe more stages of general application.

3. Phylogenetic theories of embryology

Until fairly recently, the main theoretical concern of embryologists has been to find a guiding principle which would allow them to arrange the enormous mass of descriptions of developmental changes into some sort of orderly whole. The chief such principle was found in the theory of evolution. Long before Darwin, at a time when the idea of evolution was little more than a nebulous speculation, Meckel suggested (about 1810) that a developing embryo of a ‘higher’ form of animal passes through a series of stages which represent the adults of the ‘lower’ forms ancestral to it. For instance at one stage the embryo bird has gill-slits, structures which of course are present and have a function in adult fish but disappear in the bird before the adult stage is reached. Fairly shortly after, the improvement of microscopes made it possible for von Baer to show that an embryo never looks exactly like an adult of any kind. The gill-slits of a bird embryo are rather like those of a fish embryo, but only remotely resemble those of an adult fish.

As von Baer pointed out, the fact is that young stages of different species resemble each other more than older stages do, but this does not mean that the stages in the development of an animal repeat its evolutionary history. However, in spite of his commonsense, this idea of ‘recapitulation’, as it was called, was revived after Darwin had made evolution the centre of biological fashion again. Its chief exponent was Hacckel, and for some time it was taken as the guiding principle in embryology. It was sometimes argued that evolutionary change always occurs by new stages being added on at the end of development, so that the advanced animal goes through the embryonic stages of its ancestors, perhaps in an accelerated and shortened form, then goes on a step or two further. But it was eventually borne in on embryologists that von Baer had been right (cf. de Beer 1951). And as they came to reflect on the causal mechanisms underlying embryonic development, it became clear that it is only to be expected that evolutionary alterations are much more likely to affect the later stages of development, when comparatively minor features are being formed, and to leave intact the earlier steps on which all the later stages must depend. As a matter of fact, in their very earliest stages the embryos of different types of animals are rather radically different. It is at an intermediate period, early but not right at the beginning, that embryos are most alike; probably because this is the time at which the basic structure of the animal is being rapidly laid down, and it is very difficult for evolution to alter anything at such a crucial period without throwing everything into confusion.

It is, moreover, not true that an evolutionary advance always involves the addition of something new to the original course of development. In general it consists rather in a modification of the later stages in development than in an addition to them. And there are several instances in which the evolutionary novelty has been produced by arresting development at an earlier stage than previously, so that the juvenile form of the ancestor becomes the adult of the descendant. In some respects, this has probably happened in the evolution of man; the human adult has many features which remind one of the young of apes (e.g. in the large skull with the sutures between the bones closing very late, the form of the teeth, the hairlessness of the skin, etc.). It has been argued, with perhaps less plausibility, that the ancestors of the whole phylum of vertebrates are to be found in the larval forms of echinoderms. ;

The type of analogical thinking which leads to theories that development is based on the recapitulation of ancestral stages or the like no longer seems at all convincing or even very interesting to biologists. Our interests have been awakened by the possibility of an analysis of development in causal terms; and it is in this field that modern embryology secks for its guiding principles. Recapitulation, in all the forms in which it occurs, remains an important phenomenon, but it appears nowadays as a series of problems for evolutionary theory to discuss rather than as an explanation of developmental processes.

4. The mechanisms of development

During the first phases in the study of a subject, all the available resources have usually to be concentrated on the task of providing a thorough scientific description of the phenomena involved. Embryology remained in this condition until about the end of the nineteenth century, when the first serious attempts were made to investigate the causal processes by which developmental changes are brought about. The leader in this endeavour was Wilhelm Roux, who coined the title ‘Entwicklungsmechanik’ for such studies. This word is still commonly employed in German. Its literal translation in English is “developmental mechanics’, a phrase which is not only rather long and clumsy as the name of a branch of science, but which carries a perhaps unfortunate suggestion that only machine-like, physical processes are being envisaged. Another rather awkward phrase, “experimental embryology’, is often used in English in its place. Perhaps the most satisfactory expression would be ‘epigenctics’. This is derived from the Greek word epigenesis, which Aristotle used for the theory that development is brought about through a series of causal interactions between the various parts; it also reminds one that genetic factors are among the most important determinants of development. It is, however, not yet in common use.

Since the beginning of this century, the experimental study of development has been steadily growing in importance, and is now just as indispensable a part of the science of embryology as is the purely descriptive part. Before we proceed to the detailed discussion of different types of embryos, it will be as well to give a general survey of the experimental tesults in as broad an outline as the summary description of the successive stages of development in the last section.

The study of epigenetic processes has been carried out by two radically different methods; those of experimental embryology proper, which involve interference with embryos by surgical means, or by treatment with chemical or physical agents and so on; and those of developmental genetics, in which the embryos are ‘experimented on’ by controlling the genetic constitution of the gametes from which they arise. These two lines of approach have led to two bodies of knowledge which are as yet only somewhat imperfectly brought into relation with each other, and in the outline given below it will be convenient to treat them separately. Another point to which attention should be drawn is the fact that the attempt to understand a previously unknown causal system is nearly always a slow process. In most causal sciences, and certainly in causal embryology, a long period of investigation is necessarily devoted to discovering the general nature of the causal systems involved, and only after this endeavour has made considerable progress is it possible to get down to the concrete details of how the various mechanisms work. As will be shown below, most of the theories of experimental embryology do not attempt to do more than describe the kind of system which is operating; it is only in quite recent times, and then only in a few instances, that one can begin to envisage the specific chemical reactions or physical forces concerned.

There are three basic types of phenomena which occur during embryonic development, and for which a causal science has to attempt to find some explanation. The first is the gradual change in the nature of a mass of living matter, which may consist of a part of a cell or more usually of a group of many cells. For instance, we see the columnar epithelial cells of the early neural plate gradually assume the characteristic appearance of the central nervous system, with its elaborate arrangements of nerve fibres; or the roughly cuboidal cells of the somites become elongated and filled with myosin until they are recognisable as muscle fibres. Such phenomena may be called ‘histological differentiation’; and it is most correct, indeed, to reserve the word ‘differentiation’ for changes of this kind even when it is used without qualification.

A second type of phenomenon is the arising of differences between the various parts of the embryo. Soon after fertilisation we may be able to recognise only two or three different regions, while at a later stage there will be many more individually characterised organs. Again within any one organ, such as the neural system, there are at first only a few distinct sub-units, in contrast to the numerous parts into which it becomes naturally divided later on (the fore-, mid-, and hind-brain, the spinal column, etc.). This phenomenon might be referred to by the expression ‘regional differentiation’, but actually that is usually, and better, employed to indicate the type of histological differentiation characteristic of one region (say the forebrain) when it is contrasted with that of some other region (such as the spinal column). The arising of differences between the spatial parts of the zygote is, somewhat more commonly, spoken of as ‘segregation’ (or ‘Sonderung’ in German), words whose main drawback is that they tend to suggest a particular mechanism for the process, namely a sorting out into two separate positions of materials which were originally mingled. The word ‘regionalisation’ is also used as another name for the process, and is perhaps preferable, as being more neutral in its implications.

The third basic type of process is the moulding of a mass of tissue (or, in Protozoa, of a part of the cell) into a coherent structure which is recognised as having some unitary character of its own, which is usually acknowledged by giving it a name as an anatomical organ. Thus the neural plate does not merely undergo histological differentiation and regionalisation, to give separate masses of forebrain tissue, midbrain tissue and hindbrain tissue, but also becomes moulded into the characteristic shapes of these organs. The forming of a mass of cells into a new shape is known as ‘morphogenesis’. In the abstract, one can conceive of it as occurring quite by itself, without any accompanying histological differentiation or regionalisation. But it is only rarely, in simple organisms such as Myxomiycetes or in special situations such as cells growing in tissue culture, that this happens. Much more usually, the morphogenesis of an organ is accompanied by tissue differentiation and often by the appearance of distinct spatial sub-units (regionalisation). For such complex processes, when we wish to emphasise morphogenesis as the main component, the name ‘individuation’ has been proposed.

It will be realised, of course, that in the actual phenomena of embryonic development, changes of all these three types are usually closely interwoven with one another. It is true that in experimental situations histological differentiation can occur with no regionalisation and very little, if any, individuation. But regionalisation is nearly always accompanied by some individuation, since the newly appearing regions are normally related to one another in some definite pattern. And we have already noticed that morphogenesis by itself is something of a rarity. Nevertheless it is important to disentangle them from each other, since each requires a different category of explanation. Differentiation could be a purely chemical process, involving nothing more than changes iu substances (which, however, might exist in larger particles than the molecules of conventional chemistry, for instance in cell granules, mitochondria, etc.). Regionalisation, on the other hand, involves some references to a spatial framework; it requires at least physico-chemical notions, such as diffusion, crystallisation or the like. Finally, the moulding of a mass of material into a shape, as in morphogenesis, can only be brought about by the operation of forces, and thus requires discussion in terms of physics.

It is only in recent years, as our understanding has increased, that the distinction between these types of phenomena has become important for experimental embryology. The greater part of the subject has been developed in terms of more loosely defined notions, which have in practice been closer to the idea of differentiation than to the other two concepts. For instance, experimentalists have attempted to discover the factors which bring about the development of the gut from the lower end of an echinoderm egg, or that of a neural plate from a certain region of a frog’s egg. Both these developments actually involve some regionalisation and individuation; but in the main the experiments have not been concerned with finding out what forces, arising from what sources, push the gut into the interior or fold the neural plate into its characteristic shape. Far more, the point has been to discover how the gut-developing region comes to differ from the parts which develop into something else, or from tissues which cannot develop at all. It is only after we have got at least some inkling of an answer to this problem of differentiation that we can proceed to tackle the other aspects of the processes which go on under our eyes. It will be more appropriate to postpone a discussion of the mechanisms of regionalisation and individuation until more of the facts have been presented (see Waddington1956_20Chapter XX), but it may be helpful to give here some indication of the general nature of the ideas which have developed concerning differentiation in the rather broad sense of that term which has just been mentioned.

Differentiation itself can be regarded as occurring in two phases. At an early stage during the development of any given region of the egg, its future fate becomes more or less fixed, so that it can only be altered within a narrow range by any known experimental means; thereafter, that region will always develop into one fairly definite end-product, provided of course that the conditions are such that it can develop at all. The process vy which this fixity of end-result is brought about is spoken of as the process of determination. After it has occurred there follows a long series of events which gradually transform the cells into this adult form. These are the changes which are most usually referred to when the word differentiation is used in a rather restricted sense. During determination something occurs which decides which, out of a number of possible types of development, will actually be realised; during the later phases of differentiation, this realisation comes to pass. The most important agents controlling development are, we shall argue in detail later, the genes in the nucleus. Determination is the process of bringing into operation one or another set of gene-activities; later differentiation is the result of these activities. Most embryological work has concentrated on the problem of determination, since it has seemed more important, and perhaps easier, to discover how the genes are brought into activity than to study the detailed course of the processes which they control. The idea of determination has therefore become one of the most fundamental in embryology. Recently, however, interest in the later stages of differentiation has been increasing, with the application of new methods, such as biochemical or immunological techniques for following the way in which specific substances increase in concentration.

The notion of determination is to some extent a relative one. It is defined in the first place experimentally, in that the part of the egg is said to be determined when we do not know any way of altering its later development, and of course it is always possible that new experimental methods will succeed where old ones fail. We can thus imagine a part being apparently determined in relation to one sort of experiment, but not yet determined in relation to some other. Moreover, there is the question of how specific is the end-result. For instance, a part may be ‘determined’ as eye, since it will always develop into eye whenever it can develop at all; but there may still be some possibility of controlling which part of the eye it will form (e.g. retina, tapetum, lens, etc.). Usually, in fact, a tissue gradually becomes more and more precisely determined in a series of steps as its development proceeds. Even when it has become as fully determined as it ever does, it may still have a certain restricted range of possible states which it may assume under different environmental conditions. Thus if cells from various organs of the vertebrate body are grown in tissue culture, they often lose many of their obvious visible characteristics and present an apparently ‘undifferentiated’ appearance (cf. Willmer 1935). They tend, in fact, to take on one or other of three basic cell forms, the fibroblastic, the epithelial or the wandering-cell types (Fig. 1.2). But when their powers of differentiation are tested by grafting them back into the body or otherwise, it is found that they are actually still as narrowly restricted as they were originally. The various alterations which the cells have undergone have not changed their essential nature, but are merely superficial reactions to different environmental conditions. They are usually known as ‘modulations’ (Weiss 1939). One of the most extreme examples has recently been described by Fell and Mellanby (1953); high vitamin A content in the medium causes chick embryonic skin to differentiate in tissue culture into mucus-secreting, often ciliated, epithelium instead of a squamous keratinising type. If the tissue is transferred back into a normal medium the new cells which develop are of the normal squamous kind.

In a few cases, processes which must be considered to be true “determinations’, and not mere ‘modulations’, may later be annulled. For instance, the parts of an carly ascidian embryo have only restricted possibilities of differentiation open to them and may be considered to be highly determined; but the adult animal has considerable powers of regeneration, which demand a much greater flexibility than the embryo has at its disposal. Such phenomena are not very common in the animal world. They emphasise the fact that, strictly speaking, a given process of determination occurs only in respect of the epigenetic situation at one particular stage of development. When we say, for example, that the ectoderm of a newt neurula has been determined either to become neural tissue or to become epidermis, we mean that a choice which was open to it during gastrulation has been settled one way or the other. This need not imply anything about choices which may become open to the material at some much later period in its history, for instance whether it will differentiate in one way or another during regeneration in the larval stage. In point of fact, it is only in a few special cases that any difficulty arises in this connection, but it is as well to bear the point in mind.


FIGURE 1.2 The three basic configurations which cells assume in tissue culture. Upper left, epithelial; lower left, wandering cells; right, fibroblasts. (From Willmer 1954.)


But even though the word ‘determination’ may, for such reasons, often need qualification to render it fully precise, the notion still remains extremely important. It enables us to deal with the fact that the fundamental causal happenings which control the course of development usually occur long before they can be visibly recognised. They are, as might be expected, chemical processes, not immediately detectable by the microscope, and at present only to be discovered by testing to see whether the developmental fate of the tissue can still be altered or not.

In very broad outline, one may say that experimental embryology has discovered three main types of mechanism which bring about determination. These are:

(1) Ooplasmic segregation

The different regions of the cytoplasm of the egg may have specific properties, so that a particular region can only develop in one way. Such regions are spoken of as ooplasms; an older name was ‘organ-forming substances’. The actual process of development depends on the occurrence of some sort of interaction between the cytoplasm and a nucleus which will eventually arrive in the region during the course of cleavage; but it is the cytoplasm which determines the type of development. In some eggs (for instance, ascidians or spirally cleaving eggs) there may be several such substances; in others (for instance, Amphibia) there may be only one. Again the cytoplasmic regions may be precisely localised, with sharp boundaries between them, or they may shade off into one another (as in echinoderms); in the latter case, this type of mechanism grades into the ‘field’ type mentioned under (3). The main questions about such ooplasms are, firstly, the reasons which cause them to be segregated into different parts of the egg, and secondly, the nature of the interactions between them and the nuclei.

(2) Evocation

Two neighbouring parts of an egg or embryo may react with one another, in such a way as to change the capacity for development of one, or perhaps sometimes of both, of the reactants. Processes of this kind usually take place after the period of cleavage, when the shiftings and foldings of gastrulation bring together parts of the embryo which were previously separated. By interactions between parts which have newly come together, the composition of the embryo gradually increases in complexity. Thus a region which has been determined very early, for instance, by an ooplasmic segregation, may be brought into contact with an as yet undetermined part, and exert some influence which causes that part to develop into some definite type of tissue. This type of process plays a particularly important role in vertebrates. For example, in amphibia there is an ooplasmic segregation of the so-called ‘grey crescent’ soon after fertilisation, which enables that region to develop into mesoderm; when during gastrulation, this future mesoderm is brought into contact with part of the ectoderm, it causes the latter to develop into neural tissue. In such cases the part which exerts a stimulus and thus causes the other reactant to develop into some tissue, say A, is said to ‘evocate’ A.

(3) Field action

In very many embryological processes, the development of any given point in a region of the egg depends on its relations with other nearby points or on its position within the region as a whole. For instance, if, at the beginning of gastrulation in the amphibia, a small piece of the mesoderm is cut out, rotated through 180° and replaced, a perfectly normal mesoderm may still be formed; the development of the rotated piece has been brought into line with its surroundings. Again, if a large part, or even half of the mesoderm is removed, the development of each point is modified in relation to its position within the total amount which is still left, so that again a normal embryo is formed. Such happenings are spoken of as ‘field phenomena’. The reference of this name is to physical field theories, such as those of magnetism, gravitation and so on. The implication is not, of course, that these physical forces are operating, but merely that the biological events have the same general character as the physical ones; in both cases there must be some activity spread throughout the whole region occupied by the field, and distributed in an orderly graded manner, so that in some parts the activity is strong, in others weak, with intermediate strengths between.

In some ways, field properties are complementary to those involved in ooplasmic segregation. In the latter we are confronted with a small number of differences, usually sharply distinct from one another and each confined to a particular region; in the former with graded differences in some property which spreads throughout the whole of a wide area. From another point of view, the notion of fields is closely connected with that of evocation, since when we say that the development of one point in the field is dependent on its relations with its neighbours, we must imply that those neighbours influence it in a way somewhat similar to that involved in evocation. In fact, one might conclude that ooplasmic segregation and evocation are the processes which occur in those aspects of development which involve sharp and clear-cut differences, such as the formation of different types of tissue, while field phenomena are found when the differences are blurred and intergrading as they are between the various parts of a single harmonious organ. This gives one hope that eventually it will be possible to see all three types of mechanism as mere variants of some more general type; but it is still too soon to attempt to do that, at any rate in an elementary discussion. (For a further discussion of embryonic fields, see the Appendix to this Chapter.)

It is probable that in every kind of egg, all these three types of process occur, although in some of them one type will predominate, in others another. Moreover, at one and the same time in embryonic development, processes of different types may be proceeding together. For instance, at the time of gastrulation in the Amphibia, the future mesoderm interacts with the ectoderm with which it is being brought into contact, and evocates neural tissue from it; but at the same time a field process is operating, by which not only is the mesoderm moulded into a full set of organs, but the newly evocated neural tissue is brought into the system too, so that a complete and harmonious embryo results. The name ‘induction’ is used for the whole of this complex process, of which evocation and field phenomena are separate aspects.

During the twenties and thirties the ideas sketched above were a sufficient guide to lead embryological research into ever new territories; and there are still many areas of the unknown to which they can unlock the doors. But during the last decade or so it has become increasingly clear that something further is required. The time has come to find some point of view which will suggest methods of attacking the problems of the nature of the interactions between ooplasms and nuclei, and between inducing and induced tissues or the different parts of a field. Broadly speaking, two main new approaches are being developed at the present time; one which is biochemical and cell-physiological, another which is genetical. The former is the more direct derivative of previous embryological thought. It seeks to identify and study the biochemical processes which play a crucial role in determination and differentiation, and to discover the nature and functions of intracellular structures which are important in this connection. Examples are the study of the physiology of organiser action (p. 193 et seq.), and the investigations on the biochemistry of the gradients in sca-urchin eggs and the role of the mitochondria.


Very important advances are, and will undoubtedly continue to be, made by these methods. The more strictly biochemical approach, however, encounters the difficulty that many of the happenings in a developing cell are probably related more closely to its maintenance as a living concern than to its determination and differentiation. It seems unlikely that we can hope to obtain anything like a satisfactory understanding of development in biochemical terms until we can comprehend the whole working of the cell, as regards maintenance as well as change. This consideration leads me personally to the opinion, which is by no means the most fashionable one, that it is premature to look to biochemistry to provide the main framework of ideas for embryology.

There is another approach which still requires discussion: that derived from the genetical fact that the character of differentiated organs and tissues is controlled by genes. Most people are willing to admit the relevance of this to embryology, but a study of recent books and discussionsymposia will show that in practice the contribution of genetics to embryological thought is still rather tenuous. This is in the main due to the fact that developmental genetics has been studied chiefly by people whose interests were primarily genetical, and who have posed the question: How does a given gene operate, what is the connection between a certain nucleo-protein constituent of a chromosome and some event in the cell containing it? This is obviously a fundamental question in its own right. But the progress towards an answer to it has arrived so far at little more than the statement that a change in a gene often affects the activity of a cellular enzyme or other complex molecule. From an embryological point of view, such a conclusion is somewhat trite. For embryology the question should be turned upside down; not, how does a gene operate, but how is a developing tissue affected by the genotype of the cells? We already have an answer to this which goes far enough beyond the commonplace to make a considerable difference to our whole outlook on embryological problems.

Let us therefore turn to sketch, in equally bold outline, the kind of information which has been acquired by the genetical methods of analysing development. This can be summarised as follows:

(1) There is no reason to suppose that there is any category of developmental processes which is not ultimately controlled by genes. Many of the older authors suggested that genes affect only the details of an animal’s structure while the broad outlines of it were dependent on something else. This idea contained a certain germ of truth in so far as the basic plan of the animal body is laid down in the ooplasmic segregations in the fertilised egg; but we now know of cases which show that the pattern of these segregations is itself influenced by the genes in the maternal ovary in which the egg was formed (see p. 43). The genes can therefore be regarded as the ultimate controllers of the whole range of developmental processes.

(2) It is usually held that any given gene only produces one specific immediate effect, although of course from this many secondary consequences may eventually follow in later development; and the theory is often carried a step further by the suggestion that this primary action of the gene is to influence the production of a corresponding enzyme. There is no doubt that much very beautiful work has recently revealed many genes each of which does influence the formation of a particular enzyme. But there is no very compelling reason to suppose that they do so in a single step, and that this is their primary action; nor can it be shown that all genes influence enzymes; and again it has not been demonstrated that a gene cannot have more than one primary activity, for instance by reacting with different substrates. Indeed, in the present state of our ignorance about developmental processes, it makes very little difference to our general understanding which of these many possibilities we suppose to be true. Any single gene is such a comparatively minor element in the whole complex process of the formation of a tissue or an organ that the general character of its primary action has little relevance at the present time.

(3) Genetical analysis of well-studied animals, such as Drosophila, has shown that each developmental process is influenced by very many genes. There must be many more ingredient clements in a developmental process than might be guessed at first sight. We cannot, for instance, hope to give a full account of the development of a nerve cell simply in terms of the synthesis of a single specific nerve protein by a system containing only one or a small number of kinds of molecules; we shall always be dealing with complex systems containing at least a few tens of different active substances.

(4) Genetic studies reinforce an important general conclusion which can also be drawn from purely embryological considerations; namely that the reactions between the many substances concerned in a developmental process are interlocked so that they become partially selfcompensating. That is to say, slight changes can be made to the system without producing any effect on the end-result. For instance, most genes show some degree of dominance, which means that when one dominant allele is substituted by a recessive one, little or no difference is made to the animal which develops. Embryologically, we see the same type of phenomenon when it is found that a normal organ can be formed even if we remove part of the tissue from which it would normally develop; or when we notice that embryonic cells usually develop either into one definite tissue (say liver) or into another (such as kidney) but not often into intermediates. The situation has been described by saying that development is ‘canalised’ (Waddington 19402), that is, that there are only a certain number of defined channels along which the developmental processes can go; and it must be remembered that each course of development involves complex processes in which many different genes are concerned.

(s) It is obviously not the case that all genes are being equally effective in all cells of the organism; if this were so, there could be no regional differentiation. We must suppose that a group of cells follows one particular canalised process of development because one of the possible combinations of gene-controlled processes is set going, while in another group a different set of activities occurs. It is in the investigation of how this differential activation of sets of genes is brought about that the genetical and embryological viewpoints are coming closest together at the present time. We have seen that experimental embryology has developed one set of ideas about such matters; there may be a segregation of ooplasms which can react differently with the nuclei which move into them, or there may be interactions between neighbouring tissues which are sharply distinct in character (in evocation) or only quantitatively different (in field action). It is seldom, in these embryological investigations, that the genes enter explicitly into the picture, but we are dealing with the activation of different pathways of development, and these we know, on general grounds, to be ultimately under genetical control. It is, then, only a difference in the nature of the material being studied, and the techniques available, which distinguishes such work from genetical investigation into the way in which alterations in the cytoplasm or the presence or absence of certain substances in the external medium may stimulate or inhibit the operation of particular genes. This problem, is perhaps, worthy of being called the focus of present-day analytical embryology; it is discussed at some length in Chapter XVI.

There are, of course, many other principles of more or less restricted validity, which have emerged from developmental studies, but those listed above form the main body of theory which can be generally applied throughout the whole field of embryology. When one reflects on the character of these principles one realises that experimental embryology has as yet hardly reached the stage of being able to investigate the actual causal mechanisms which bring about developmental changes. For the most part, it is still concerned to discover and describe the general nature of the system which is in operation on any particular embryo. If one says that the early development of the molluscan egg is mainly dependent on ooplasmic segregations, that does not tell us anything of the causes which bring about the segregation, or of how the various ooplasms cause the appearance of the specific characteristics of the organs to which they give rise; what we have done is to describe a type of system, but we are still unable to point to particular causes and their particular effects. The same is true when we attribute the development of the echinoderm egg to a system of gradients or fields. It is only in connection with evocation phenomena that we begin to attain any real experimental control over important developmental events, since where evocation comes into play, we can switch the development of a piece of tissue one way or another by placing it either near to or far from the source’ of the evocating stimulus. In this connection, then, we are already in contact with a basic causal system and can hope to go beyond finding out the general nature of the system to the crucial step of discovering what it actually is in detail. Unfortunately, as we shall see in Chapter X, it has turned out to be casier to sce this bird than to put salt on its tail.

From the genetical side, also, we are as yet only just approaching the actual causal systems. We know somethi g about the kinds of things genes, or groups of genes, do; but we still want to know exactly what some one definite gene does and how it produces its effects. It is only in a few cases that we can control the activities of genes, and it is not until we can do so, that we can hope to discover much about them. Again, €vocator reactions provide one example; by the presence or absence of the stimulus we can bring into play one or another set of gene-controlled processes; but the genetic variants are not available in any of our laboratory stocks which could make it possible to analyse this situation genetically. The other instances in which it is possible to determine experimentally whether a gene shall be active or not occur in lower and more or less undifferentiated organisms (for instance in the control of immunological Properties in the protozoan Paramecium, or the formation of adaptive enzymes in bacteria and yeasts). From such cases we cannot learn much about the precise mechanisms of differentiation, but we can find some interesting general guidance.

It is in the analysis, by developmental genetical methods, of the formation of certain particular chemical substances, that we have so far come nearest to a full understanding of any developmental process. We know a great deal, for instance, about the development of the pigments in eyes of the fly Drosophila; not only the genes that affect it, but also the chemical nature of some of the most important changes which those genes produce. Unfortunately, the substances about which we have such detailed knowledge are comparatively trivial ones; we do not have such information for anything as complex as a protein, let alone for any particular type of cell or tissue.

APPENDIX THE CONCEPT OF EMBRYONIC FIELDS

The field concept has been widely used in some recent discussions of development, notably by authors such as Huxley and de Beer (1934), Weiss (1939) and Lehmann (1945). It is, however, rather difficult to make clear exactly what is meant by it and unless the term is given a fairly precise meaning it is only too easy to use it as a sort of ‘joker’ by which almost anything can be explained (see review of Huxley’s and de Beer’s book by Waddington 19346, and Needham’s discussion 1942, p. 127).

The first confusion arises from a tendency to use the work ‘field’ when all that is meant is a reference to the geographical location in which something is happening, while not implying anything about the nature of the events going on there. In such circumstances it is better to use a more neutral and clearly geographical term. For instance, in the neurula of an amphibian embryo the right forelimb will arise from a quite definite place. This should be referred to as the limb area, not as the limb field. At an earlier stage the localisation of the limb is not so precise. Experimentally it may be caused to appear anywhere within a somewhat larger region of the embryo. Needham has suggested that these larger regions may be referred to as ‘limb districts’. We may thus speak of the ‘limb district’ in an earlier embryo, meaning the whole region out of which a limb could be caused to appear, and in a later stage in which the position of the limb had been more precisely fixed we could begin to speak of limb ‘area’.

The word ‘field’ should be used only when we wish to refer to the character of the processes which go on in an area or district. By using the word we mean to imply that there are a number of processes which interact with one another in such a way that they take up definite relations to one another in space. It is easier to show what this means in a concrete example than by abstract definitions. Unfortunately there is no actual case in which the causal mechanisms of an embryological field are truly understood. It will therefore be necessary to give an imaginary example, which however will serve to show the general nature of the ideas which should be at the back of one’s mind when one uses the concept of fields.

Imagine, then, a flat expanse of tissue, as it might be ectoderm on the surface of an early embryo (Fig. 1.3). Suppose that this has the following properties: (i) that it has an anterior-posterior polarity, and that some substance B is present in a gradient with a high concentration at the posterior, sinking to a low level anteriorly; (2) that the micro-structure




FIGURE 1.3 The development of an (imaginary) embryonic field.

(i) represents a section through a flat expanse of tissue, into which oxygen is diffusing from one side, while some substance is being formed at A (concentration indicated by dotted line) and diffusing outwards; (2) and (3) show sections through which the field which is developing as explained in the text.

of the tissue is such that diffusion is faster within the plane of the tissue than it is vertically through the thickness; (3) that over the whole lower surface of the tissue, there is some precursor substance a which can be autocatalytically converted into an active substance A. Now imagine that at some particular point in the region, this conversion begins to occur, and A to be formed (this position may be determined by the normal surroundings of the tissue, or by some experimental means). A will now diffuse slowly upwards through the thickness of the tissue, and more rapidly within the tissue away from its point of origin in all directions. Let us further suppose that the activity of A consists in causing the tissue containing it to become heaped together into a thicker layer, and also that its formation is inhibited by oxygen diffusing into the tissue from the outer surface. Then where A first starts to form, we shall have a heaping up of tissue, leading to a thinning of the surrounding ring of material; and in this surrounding ring oxygen will penetrate a greater relative thickness of the material, so that the formation of A is reduced; while outside the ring a higher concentration of A will be able to build up. Thus we shall have a system consisting of a central knob of tissue surrounded by a groove outside which is a thickened ring of lower elevation than the knob. This is already a structure which could be considered as an organ, with a definite characteristic shape. We may refer to it as the organ X. If the substance A interacts with another substance B which is present in a graded. concentration in the tissue, the shape of the organ will not be radially symmetrical round the point of origin, but will be bilaterally symmetrical.

Now it is in situations such as this that embryologists have often used the expression ‘the X field’. They have meant two rather different things by it. The most valid use of the term refers to the situation within the region around the point at which A is being formed. Here we have a series of processes—of the appearance of A, its diffusion, its reaction with oxygen and with substance B, the heaping up of the tissue in one place and its thinning nearby—all of which interact on each other in a way which results in the region developing through a definite series of steps into a well-defined end-result, the organ X. The term ‘field’ is used to emphasise the co-ordinated and integrated character of the whole complex of processes. When it is used in connection with the formation of a definite organ with a characteristic individual shape, the term can be made more precise by qualifying it as an ‘individuation field’ (Waddington and Schmidt 1933).

The word ‘field’ is also sometimes used, in a rather less legitimate manner, to refer to the conditions within such a region of tissue before the point at which A will be formed is precisely localised. For instance, in the flank of an early amphibian embryo, the formation of a limb can be induced by implanting various substances into the mesoderm (p. 273). One may come across such a phrase as ‘the forelimb field extends from about the second segment to about the tenth, reaching a maximum intensity in segments three to six’. Here we are dealing, not with the individuation field, which is confined to the area in which a limb is actually developing and the immediate neighbourhood of this, but with the large region in which there are the preconditions necessary for the appearance on the individuation field. Such a region could better be referred to as the ‘region of competence’ for the organ (Waddington 19346) or the organ ‘district’ (Needham 1942). But the fact that its properties are not usually equal throughout, but are graded from a high value in the centre to low values at the periphery, has frequently tempted people to go on using the word ‘field’ for it; and they probably will continue to do so. Not much harm is done by such a usage if one stops to think what one is doing.

As development proceeds, a region or district of competence gradually turns into an individuation field. One can show the character of the changes that occur in a diagram such as that of Fig. 1.4. At an early stage, any part of the district can be caused to develop into the organ by bringing the concentration of some activity A above the threshold; but the ease of doing this will be distributed in a graded way. Once the threshold has been reached, and the organ begins to be formed, its individuation field gradually extends till at its maximum it covers a rather larger area than that out of which the organ is produced. In the peripheral regions, the effect of the field is to suppress any tendency for a second organ to be formed. As the formation of the organ proceeds, the extent of the field usually contracts again until it is confined to the organ itself; and in the meantime the competence of the outlying parts of the district disappears, so that the possibility of another organ appearing lapses. In some of the lower animals, however, in which regeneration is possible throughout life, the field remains in an extended form, controlling the competence of the peripheral parts of the district and suppressing its ability to produce supernumerary organs.



4 FIGURE 1.4 The development of a district of competence into an individuation field.

The diagrams show a longitudinal section through a region of a developing embryo. At the earliest stage (1) the precursor (A) of a certain organ has not yet reached the threshold anywhere; this is the stage of a ‘district of competence’. In (2) the concentration of the active substance A has reached the threshold and an individuation field for the organ is beginning to arise. In (3) the individuation field extends outside the area in which the organ will actually appear, which is dotted. The peripheral parts of the individuation field depress the level of A. This is the stage which persists in lower forms in which regeneration is possible. In (4) the individuation field has contracted until it is confined to the area of the developing organ; meanwhile the substance A has disappeared in the outer parts of the region, partly owing to the suppressive action of the individuation field and partly as a simple result of the progress of development.



FIGURE 1.5 Behaviour of active fields.

(1) If a field is cut in half, each portion will develop into a complete unit; they may each retain their original polarity (above) or one may be the mirror image of the other (below). (2) If two fields are brought together and allowed to fuse, they form a single field (most easily if their polarity is the same). (3) Ifa central (a) or peripheral (b) region is removed from a field, the remainder will still form a complete unit (above), while the small isolates may each also form complete fields (below).


When an individuation field is active, it shows many properties which remind one of the behaviour of magnetic or other physical fields (Fig. 1.5). For instance, if a field is cut in two each half may reconstitute a complete field, so that two whole organs are developed. These are often mirror images of one another. On the other hand, if two fields are brought together and allowed to fuse, they may rearrange themselves into a single field. Again, if a part of a field, either central or peripheral, is removed, the remainder may compensate for the defect and become complete again, while the isolated part can often become modified into a small but com plete field.

Suggested Reading

The main reference books are: for descriptive embryology of invertebrates, Heider (1936); of vertebrates, Nelsen (1953); for experimental work, Schleip (1929), Lehmann (1945, echinoderms and Amphibia only); for biochemical aspects, Needham (1931, 1942), Brachet (1944). Two of the most important forerunners of modern embryology were Driesch (see 1929) and Roux (whose views are discussed in Needham 1936a and Russell 1930).

There have been several general conferences and colloquia on embryology in recent years; those published by the Society for Experimental Biology (‘Growth’, Second Symposium, 1948), the New York Academy of Science (Annals, Volume 49) and in the Revue Suisse de Zoologie, Volume 57 (Supplement), are particularly worth reading.

For the relations between embryology and phylogeny, de Beer (1951).


   Principles of Embryology (1956): Part 1 - 1 The Science of Embryology | 2 The Gametes | 3 Fertilisation | 4 Cleavage | 5 The Echinoderms | 6 Spirally Cleaving Eggs | 7 The Ascidians and Amphioxus | 8 The Insects | 9 The Vertebrates: The Amphibia and Birds | 10 The Epigenetics of the Embryonic Axis | 11 Embryo Formation in Other Groups of Vertebrates | 12 Organ Development in Vertebrates | 13 Growth | 14 Regeneration | 15 The Role of Genes in the Epigenetic System | 16 The Activation of Genes by the Cytoplasm | 17 The Synthesis of New Substances | 18 Plasmagenes | 19 The Differentiating System | 20 Individuation - The Formation of Pattern and Shape | References
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