^ ^*^^1?"5h*^^ k,SK. •V"*"* PRINCIPLES OF EMBRYOLOGY BY C. H. WADDINGTON How Animals Develop Introduction to Modem Genetics PRINCIPLES OF EMBRYOLOGY BY C. H. WADDINGTON SC.D., F.R.S. Professor of Animal Genetics University of Edinburgh vJSo I ISC WITH .186 FIGURES ^tmmmm-^ LIBRARY WOOOS HOLE, MASS. W. H. 0. I. NEW YORK THE MACMILLAN COMPANY FIRST PUBLISHED IN I956 This book is copyright under the Berne Convention. Apart from any fair dealing for the purposes of private study, research, criticism or review, as permitted under the Copyright Act igii, no portion may be reproduced by any process without written permission. Enquiry should be made to the publisher PRINTED IN GREAT BRITAIN PREFACE IN WRITING this book I have had three aims in mind. I have tried to ex- pound a picture of embryology, which has been formed during a quarter of a century's work, and to do so in a form sufficiently factual and system- atic to be useful as a textbook for students specialising in that subject, or in the allied fields of genetics or experimental zoology. At the same time, I have attempted to meet the needs of research workers in other branches of biology who wish to fmd out what is going on in the study of develop- ment at the present time. Embryology grew up as a branch of comparative anatomy; and when the science is referred to without qualification, even to-day most biologists probably think first of a descriptive account of developmental changes in anatomy and histology. But there is, of course, by now a very large body of data relating to the causal analysis of development. This is often re- garded as a separate corpus of knowledge, referred to not as 'embryology' but as 'experimental embryology'. A few decades ago, phylogeny and the evolutionary aspects of comparative anatomy constituted the core of animal biology, and it was not unjustified for the descriptive approach to development to be accorded the title 'embryology' tout simple. But now the situation seems to me to be different. The part of our subject which is of prime interest as a facet of general biology is that which deals with causal analysis, and if anyone claims to have studied embryology, this is the part which we ought to expect him to know about. I have therefore distributed the weight in this book in a manner quite different to that usual in textbooks of embryology, with more emphasis on the experi- mental and less on the descriptive approach, hi fact, of the latter I have provided only the bare minimum which suffices to make the experimental work comprehensible. This book is, however, not intended to be for most students their first contact with embryology, but rather to serve the needs of their later university years; and it is to be expected that most users of it will have made some preliminary acquaintance with the anatomical facts, either in practical class work or through one of the many elementary texts which exist. Perhaps the ideal previous reading would be Barth's excellent Embryology, which has the advantage of providing not only a fuller descriptive account, but also a very stimulating introduction to the experimental analysis. In surveying such a wide field as embryology, within a compass that can be used as a text by students, a considerable amount of selection has to be exercised. It is natural, and indeed probably desirable, that an author should devote most attention to those aspects of the subject on which he has himself worked. I am conscious that I have given more space to the VI PREFACE amphibia, birds and Drosophila, and less, say, to the echinoderms and the problems of fertiHsation, than some other authors might have done. I think, however, that it is not merely a bee in my personal bonnet which has led me to include in the book a considerable discussion of topics which are conventionally counted as belonging to genetics. Embryology at the present time is in a betwixt-and-between state. It can no longer be wholly satisfied to operate in terms of the 'complex components' (such as organ- isers, fields and the hke), which were discovered in the first successful experimental forays. On the other hand it is still too early to hope to find biochemical approaches which throw a general illumination on the scene. It is probably useful to try to formulate conceptional schemes in generahsed chemical terms, such as those proposed by Weiss, or that discussed in Chapter XIX; but these must be recognised as no more than very abstract guides to possible directions which our thoughts may take. We have still to work through a region of facts and theories which deal with cellular constituents ; and among this group of entities, which includes microsomes, mitochondria and such bodies, the genes (and possibly the plasmagenes) are certainly of crucial importance. It seems probable then that the most fundamental embryological theories of the immediate future will be phrased largely in terms of genes or of other bodies of a similar order of complexity; and in so far as this is true, no adequate discussion of embryology can be given without devoting a great deal of attention to the related aspects of genetics. One of the difficulties in writing a book of this kind is to decide what references to literature should be provided. Anything approaching a com- plete bibhography would be too unwieldy. I have attempted two things; to provide an introduction to modem trends of work by giving a fairly large number of citations of recent papers whose results are being quoted; and to strike a balance between giving credit to the first discoverers of various facts and ideas, and indicating the most up-to-date summaries and reviews of the different topics. I can only beg the indulgence of any of my colleagues who may feel that I have either overlooked their priority or failed to recognise the soundness of a recent summing-up. In any case, the bibhographic apparatus of such a book is inevitably a forest in which the student can only too easily lose himself I have therefore, at the end of each chapter, given a very short selection of works which are suggested as valu- able further reading, either to bring the student in contact with some of the original factual material, or to introduce him to some of the stimulating ideas which run parallel to, or even contradict, those advanced in the text. Edinburgh, July 1954 C.H.W. CONTENTS Preface page v PART ONE THE FACTS OF DEVELOPMENT I. THE SCIENCE OF EMBRYOLOGY 3 1. The place of embryology among the biological sciences 3 2. An outline of development 5 3. Phylogenetic theories of embryology 8 4. The mechanisms of development 10 Appendix. The concept of embryonic fields 23 n. THE GAMETES 29 1. Spermatogenesis 30 2. Oogenesis 31 3. Follicles and membranes 37 4. TAe morphogenetic structure of the egg 40 m. FERTILISATION 45 1. Activation 47 2. T/ie i»i/o« (>/"f/2e nuclei 50 3. Artificial parthenogenesis 51 IV. CLEAVAGE 57 1. General features 57 2. TAe pattern of cleavage and the pattern of the embryo 62 3. Differentiation ivithout cleavage 63 4. Cleavage without nuclei 64 5. The mechanism of cleavage 64 V. THE ECHINODERMS ' 79 1. Normal development 79 2. T/?e gradient system 82 3 . The dorso-ventral axis 92 VI. SPIRALLY CLEAVING EGGS 94 Vn. THE ASCIDIANS AND AMPHIOXUS IO5 Viii CONTENTS VIII. THE INSECTS Il8 1. Embryonic development Ii8 2. Experimental analysis of some types of insect development 123 3. The transformation into the adult 136 4. The determination of imaginal characters 140 IX. THE vertebrates: amphibia and birds 145 I. From the unfertilised egg to the formation of the blastula a. Amphibia b. Birds 145 154 2. 3. Gastrulation: presumptive maps The gastrulation movements a. Amphibia b. Birds 157 161 167 4- General properties of the gastrulation movements 170 X. the epigenetics of the embryonic axis 173 1. Amphibia I73 2. Birds . 181 2. Evocation and individuation 188 4. The physiology of organiser action 193 5. What occurs during evocation? 206 6. Regionally specific evocation 215 7. Competence 218 XI. embryo formation in other groups of 223 VERTEBRATES 1. Cyclostomes and other primitive fish 224 2. Teleosts 226 3. Reptiles , 234 4. Mammals 230 5. Comparative geography of the presumptive areas 242 6. Comparative causal embryology of the vertebrates 246 XII. ORGAN DEVELOPMENT IN VERTEBRATES 25O 1. The general form of the embryo 250 2. The development of the head 253 3. The gut: anterior portion 256 4. The trunk: ectodermal organs 258 5. The trunk: endodermal structures 260 6. T/je (runfe; mesodermal structures 262 7. T/je faj/ and hind part of the body 263 8. The kidneys 265 9. T/ie limbs 270 CONTENTS IX Xm. GROWTH 279 1. Overall groii'th 279 2. The relative growth of parts 287 3. Growth gradients and transformations of shape 290 XIV. REGENERATION 302 1. The origin of regeneration cells and their potentialities 303 2. Field action in regeneration a. Coelenterates 309 b. Flat worms 3I7 c. Amphibia 321 PART TWO THE FUNDAMENTAL MECHANISMS OF DEVELOPMENT XV. THE ROLE OF THE GENES IN THE EPIGENETIC 329 SYSTEM 1. Developmental pathways and their genetic control 329 2. Primary and secondary effects of genes 341 XVI. THE ACTIVATION OF THE GENES BY THE 348 CYTOPLASM 1. The effects of the cytoplasm on the nucleus 351 2. Effects on chromosomes and genes 355 3. Complete or partial inactivation of genes? 361 4. The mechanism of gene activation or inhibition • 364 XVII. THE SYNTHESIS OF NEW SUBSTANCES 3^7 1. The parts of the cell 367 2. Arguing from the gene to the substance 371 3. Arguing from the substance to the gene 376 4. Genes and enzymes 378 5. The synthesis of proteins , 381 XVIII. PLASMAGENES 3^7 1. Exogenous plasmagenes 389 2. True plasmagenes 392 3. Visible cytoplasmic particles with genetic continuity 393 4. Gene-initiated plasmagenes 39^ 5. The role of plasmagenes in differentiation 401 CONTENTS XIX. THE DIFFERENTIATING SYSTEM 406 XX. INDIVIDUATION — THE FORMATION OF PAT- 415 TERN AND SHAPE 1. Primary and secondary expressions of pattern 417 2. The origination of pattern 418 3. Some actual patterns ■ a. Drosophila wing venation 424 b. The pentadactyl limb 428 4. Morphogenesis a. Movements of isolated cells 433 b. Movements of tissues : amphibian gastrulation 437 5. Measurement of the forces and energy involved in morpho- genesis 452 6. Individuation of the central nervous system in Amphibia 455 BIBLIOGRAPHY " 4^9 AUTHOR INDEX 497 SUBJECT INDEX 503 PART ONE THE FACTS OF DEVELOPMENT CHAPTER I THE SCIENCE OF EMBRYOLOGY I. 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 devel- opment 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 pheno- mena which may also be considered as parts of endocrinology or of gene- tics. 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 consti- tute 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 life- 4 PRINCIPLES OF EMBRYOLOGY times. 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 pheno- mena 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 intellec- tually attractive. The study of evolution, which was until recently only shghtly 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 neglec- ted 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 fmd 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 httle progress can be made towards an understanding of the causal mechanisms of evolution v^dthout 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 consti- tutes 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 physio- logy, 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 fmd ourselves confronted with that most lament- THE SCIENCE OF EMBRYOLOGY 5 able 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 pro- vince 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 under- standing. New methods can usually only be appHed 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 simphfied 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 develop- ment. One can, in fact, sketch a broad outline of the early stages of devel- opment which apphes, roughly at least, to all the animal phyla. This can best be described in terms of a series of stages : Stage I. 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 em- bryology 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 apphed to the growing egg in the ovary, which are concerned in 6 PRINCIPLES OF EMBRYOLOGY 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 pro- vides 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 defm- ing 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 cor- tex-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 an- other, and we shall see that activation can happen without any union of the nuclei taking place. Stagey- 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 hlastula. 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. THE SCIENCE OF EMBRYOLOGY 7 Stage 5. Gastmlation. In a short and extremely critical period of develop- ment, 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 imier 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 blas- tulae 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 funda- mental pattern of the embryo begins to appear. In most cases, the organs wliich 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 Neural lube Somite Nolochord Lateral mesoderm Figure i.i To illustrate the basic structure of a generalised vertebrate embryo. 8 PRINCIPLES OF EMBRYOLOGY 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. I.I. 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 fmally a tube which sinks below the surface and differ- entiates 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 meso- derm is thickened and transversely segmented so that it takes the form of a series of roughly cuboidal blocks, which are knovra. 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 wiU 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 earher in the anterior end of the embryo than in the posterior. 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 1 8 10) 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, THE SCIENCE OF EMBRYOLOGY 9 Structures which of course are present and have a function in aduk 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-sHts 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, tliis idea of 'recapitulation', as it was called, was revived after Darwin had made evolution the centre of biological fashion again. Its cliief exponent was Haeckel, 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 short- ened 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 195 1). And as they came to reflect on the causal mechanisms underlying embryonic development, it became clear that it is only to be expected that evolution- ary alterations are much more likely to affect the later stages of develop- ment, when comparatively minor features are being formed, and to leave intact the earher 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 develop- ment 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 thejuvenile 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 plausi- 10 PRINCIPLES OF EMBRYOLOGY bility, 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 develop- ment 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 seeks 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 re- sources 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 pro- cesses by which developmental changes are brought about. The leader in this endeavour was Wilhelm Roux, who coined the title 'Entwicklungs- mechanik' for such studies. This word is still commonly employed in German. Its hteral 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 EngHsh in its place. Perhaps the most satisfactory expression would be 'epigenetics'. 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 develop- ment has been steadily growing in importance, and is now just as indis- pensable 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 results 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 THE SCIENCE OF EMBRYOLOGY II involve interference w^ith embryos by surgical means, or by treatment w^ith 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 v^hich 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. An- other point to which attention should be drav^oi 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 embry- onic 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 coluronar 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 fdled 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 fertihsation 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 ^orebrain) when it is contrasted with that of some other 12 PRINCIPLES OF EMBRYOLOGY 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 'regionahsation' 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 recog- nised as having some unitary character of its own, which is usually ack- nowledged by giving it a name as an anatomical organ. Thus the neural plate does not merely undergo histological differentiation and regionahsa- tion, to give separate masses of forebrain tissue, midbrain tissue and hind- brain 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 regionahsation. But it is only rarely, in simple organisms such as Myxo- mycetes 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 (regionahsation). 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 inter- woven with one another. It is true that in experimental situations histo- logical differentiation can occur with no regionahsation and very little, if any, individuation. But regionahsation 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. Never- theless 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 in substances (which, however, might exist in larger particles than the molecules of conventional chemistry, for instance in cell granules, mito- chondria, etc.). Regionahsation, on the other hand, involves some refer- ences 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 THE SCIENCE OF EMBRYOLOGY I3 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 defmed 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 ecliinoderm egg, or that of a neural plate from a certain region of a frog's egg. Both these developments actually involve some regionalis- ation 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 Chapter XX), but it may be helpful to give here some indication of the general nature of the ideas wliich 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 knov^oi 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 by which this frxity 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 wliich 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 reahsed ; during the later phases of differ- entiation, this reahsation comes to pass. The most important agents con- trolling development are, we shall argue in detail later, the genes in the nucleus. Determination is the process of bringing into operation one or 14 PRINCIPLES OF EMBRYOLOGY 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 determina- tion 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 sub- stances 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 develop- ment, and of course it is always possible that new experimental methods will succeed where old ones fail. We can thus imagine a part being appar- ently 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 con- ditions. Thus if cells from various organs of the vertebrate body are grown in tissue culture, they often lose many of their obvious visible character- istics 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 in- to 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 knovm 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. THE SCIENCE OF EMBRYOLOGY 15 In a few cases, processes which must be considered to be true 'determina- tions', and not mere 'modulations', may later be annulled. For instance, the parts of an early ascidian embryo have only restricted possibilities of differentiation open to them and may be considered to be highly deter- mined; but the adult animal has considerable powers of regeneration, which demand a much greater flexibility than the embryo has at its dis- posal. 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 Mitochond Nuclear Membra Nucleolus Fat Droplet Fat droplet Vacuole Nuclear membrane Mitochondria Nucleolus Figure 1.2 Nucleolus Nuclear membrane Fat droplets and cell granules Mitochondria 1 in division Centrosphere The three basic configurations which cells assume in tissue cukure. Upper left, epithelial; lower left, wandering cells ; right, fibroblasts. (From Willmer I954-) 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 l6 PRINCIPLES OF EMBRYOLOGY 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. But even though the word 'determination' may, for such reasons, often need quahfication to render it fully precise, the notion still remains ex- tremely 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 develop- mental 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 determina- tion. These are: (i) 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, Ampliibia) 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 THE SCIENCE OF EMBRYOLOGY I7 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 defmite type of tissue. This type of process plays a particularly important role in vertebrates. For example, in amphi- bia there is an ooplasmic segregation of the so-called 'grey crescent' soon after fertilisation, which enables that region to develop into meso- derm; 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 meso- derm 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 imphca- tion 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 num- ber of differences, usually sharply distinct from one another and each confmed 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 l8 PRINCIPLES OF EMBRYOLOGY 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 differ- ences 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 develop- ment, processes of different types may be proceeding together. For in- stance, 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 evo- cation and field phenomena are separate aspects. During the twenties and thirties the ideas sketched above were a suffi- cient 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 fmd 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 embryo- logical 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 intra-cellular 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 sea-urchin eggs and the role of the mitochondria. THE SCIENCE OF EMBRYOLOGY 19 Very important advances are, and will undoubtedly continue to be, made by these methods. The more strictly biochemical approach, how- ever, encounters the difficulty that many of the happenings in a developing cell are probably related more closely to its maintenance as a living con- cern than to its determination and differentiation. It seems unhkely that we can hope to obtain anything like a satisfactory understanding of develop- ment 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 fashion- able 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 rele- vance of this to embryology, but a study of recent books and discussion- symposia will show that in practice the contribution of genetics to embryo- logical 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 httle 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 com- monplace 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 : (i) There is no reason to suppose that there is any category of develop- mental 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 fertihsed egg ; but we now know of cases which show that the pattern of 20 PRINCIPLES OF EMBRYOLOGY 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 re- garded 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 conse- quences may eventually follow in later development; and the theory is ofi;en 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 compelhng 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 possibffities 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 elements in a developmental pro- cess 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 develop- mental process are interlocked so that they become partially self-compen- sating. 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 pheno- menon when it is found that a normal organ can be formed even if we THE SCIENCE OF EMBRYOLOGY 21 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 inter- mediates. The situation has been described by saying that development is 'canalised' (Waddington 1940^), that is, that there are only a certain num- ber 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. (5) 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 par- ticular canalised process of development because one of the possible com- binations 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 activa- tion of different pathways of development, and these we know, on general grounds, to be ultimately under genetical control. It is, then, only a differ- ence 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 ^^ PRINCIPLES OF EMBRYOLOGY 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 comiection with evocation phenomena that we begin to attain any real experimental control over important developmental events, smce where evocation comes into play, we can switch the development of a piece of tissue one way or an- other 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 o 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 easier to see 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 something about the kinds of things genes, or groups of genes, do; but we still want to know exactly what some one defmite 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 evocator 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 ^ocks which could make it possible to analyse this situation geneticaUy. The other mstances in which it is possible to determine experimentaUy whether a gene shall be active or not occur in lower and more or less undifferentiated organisms (for instance in the control of immunoloo-ical properties m the protozoan Paramecium, or the formation of adaptive enzymes m 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 forma- tion of certam particular chemical substances, that we have so far come nearest to a fuU understanding of any developmental process. We know a great deal, for mstance, about the development of the pigments in eyes of the RjDrosophila; 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 know- ■ THE SCIENCE OF EMBRYOLOGY 23 ledge 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 (i934),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 1934^, 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 some- thing 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 hmb 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 earher 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 inter- act 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 defmitions. 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 24 PRINCIPLES OF EMBRYOLOGY 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 ^^ ^' Op O2 O2 i i i i i ■M I .'""^- ~s *" , -''' ^^^-, t A / ,-v \_^^ 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 (con- centration 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 o£ 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 THE SCIENCE OF EMBRYOLOGY 2$ thickness of the material, so that the formation of A is reduced ; while outside the ring a higher concentration of yl will be able to build up. Thus we shall have a system consisting of a central knob of tissue sur- rounded 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 defmite 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^, 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 defmite 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 com- plex of processes. When it is used in connection with the formation of a defmite 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 confmed 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 26 PRINCIPLES OF EMBRYOLOGY 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. 14. At an early stage, any part of the district can be caused to develop into the organ by ^'"^^ mdividuatiort \ fuU n. n .^. I ! ' 7 ' ' Figuhe 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 (i) the precursor (A) of a certain organ has not yet reached the threshold anywhere; this is the stage of a 'district of com- petence'. 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 .4. 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 ^ 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. 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 in- dividuation field gradually extends till at its maximum it covers a rather larger area than that out of which the organ is produced, hi 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 THE SCIENCE OF EMBRYOLOGY 27 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 .y\ Figure 1.5 Behaviour of active fields. (i) 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) If a central {a) or peripheral (t) 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). competence of the peripheral parts of the district and suppressing its ability to produce supernumerary organs. 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 com- plete 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 to- gether and allowed to fuse, they may rearrange themselves into a single 28 PRINCIPLES OF EMBRYOLOGY 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, Nelscn (1953); for experimental work, Schleip (1929), Lehmann (1945, ecbinodernis and Amphibia only); for biochemical aspects, Needham (193 1, 1942), Brachet (1944). Two of the most important foreruimers of modem embryology were Driesch (see 1929) and Roux (whose views are discussed in Needham 1936a and Russell 1930). There have been several general conferences and coUoquia 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 (195 1). CHAPTER n THE GAMETES DEVELOPMENT in sexually reproducing organisms is usually considered to begin at the time when the sperm unites with the egg. But actually these two types of cells are among the most complex formed in the animal body, and themselves undergo very important processes of development before they are ready to perform their characteristic functions. The undifferen- tiated cells which will eventually give rise to them are collectively known as gametocytes, and separately as oocytes if they will form ova, or sperma- tocytes if they will form sperm. The fully differentiated cells are known as gametes. The male type are called sperm, or spermatozoa, both terms being correct and with exactly the same meaning. The female type are referred to as eggs or ova, but these two words do not mean quite the same thing. The word ovum refers strictly to the gamete-cell; and this often makes up only a part of the body known as the egg, which may include a number of membranes, layers of jelly, shell, etc., which strictly speaking lie outside the ovum, and are no part of it. Thus in the hen's egg, the ovum is only that part conventionally known as the 'yoh^'. The basic functions of the gametes are, firstly, to bring together the two nuclei contributed to the offspring by the parents, and secondly to carry out the development of the new individual until it is fully enough formed to take in its own nourishment. A good deal of preparatory differentiation is required before an ordinary cell can be fitted for either task. It is not appropriate here to discuss in any detail the preparation of the gamete-nucleus, since this subject really belongs to the alhed discipline of Genetics, and is fully described in textbooks of that subject. It is only necessary to remember that, whereas the nucleus of a normal body cell contains two of each kind of chromosome, and thus two of each kind of gene, in the gamete-nucleus these are reduced to one representative of each kind. The reduction takes place by a sequence of two divisions, known as the meiotic, or maturation divisions (the term 'reduction division' cannot strictly be apphed to either the first or second of these, but only to both together). The matured gamete-nuclei, containing only one of each sort of chromosome, are known as haploid, while the normal condition is known as diploid. ^ ^ In some organisms (many plants and a few animals) the body cells contain more than two of each kind of chromosome, and are then said to be 'polyploid' ; in this case the gametes, if they are formed at all regularly, contain half the number in the body cells, and thus more than the haploid number; but many irregularities occur in such cases. 29 30 PRINCIPLES OF EMBRYOLOGY I. Spermatogenesis In the development of sperm, the two meiotic divisions occur fairly early in the history of the spermatocyte, usually with only a short interval between them. Since there are two maturation divisions, and each division gives rise to two similar daughter cells, one spermatocyte which starts the process will eventually form four sperm. Usually they separate from one another, but in some species they remain together as a group (Fig. 2.1). Spermatogonia 2n Spe^mQ^■ocy^es ReducHon Divisions ton SPERMATIDS PERIOD OF DIFFERENTIATION SPERM ! I I I Oogonia 2n Oocyt'e PERIOD OF DIFFERENTIATION OVUM Reduchon Divisions to n Formahon oF Polar Bodies Figure 2.1 Diagram of the formation of sperm (on left) and eggs (on right). At the time when the maturation divisions occur, the spermatocytes are fairly normal-looking cells; the main differentiation by which they become transformed into sperm occurs in the haploid daughter cells. In a few groups, the fully differentiated sperm are amoeboid (e.g. some Crustacea), but in most animals they are built on roughly the same plan, consisting of three main parts; a head containing the nucleus, a middle- piece containing one or more centrosomes, and a flagellar tail which serves as an organ of motion. (For a comparative account of sperm THE GAMETES 3I morphology, seeRetzius 1902-1909.) The processes of formation of these speciaHsed parts of the cells have been the object of considerable micro- scopial study (c£ Gresson 1948) but the results have not been very clear cut, nor have different investigators always reached agreement. This is nov^adays not so surprising, since studies with the electron microscope have shown that sperm contain many structures which are well below the resolving power of the hght microscope, so that studies with the latter could not be expected to reveal the mode of their formation. The electron microscope work is still in its infancy, and again it is the case that agree- ment on the structures has not yet been reached. What is important is that this new tool has shown that the material architecture of the sperm is certainly very much more comphcated than had been suspected. An indi- cation of the degree of complexity involved may be had from Fig. 2.2, which shows the structure of the middle piece of a ram's sperm, as inter- preted by Randall and Friedlaender (1950). Even if some revision later turns out to be necessary to this picture, it is impressive to discover that such an apparently simple object can contain so many structurally distinct components arranged in such defmite and elaborate patterns. The spermatozoon is a small light cell, capable of independent move- ment. It plays the active role in fertilisation, in contrast to the immobile and passive egg. This activity is, in fact, the basic definition of a male gamete ; in some organisms the sperm is very unHke the common pattern just described, and in lower plants, for instance, there may be very Httle difference in shape between the female and male gametes ; but wherever there is a difference in activity, we say that the more active type is the male, the less active the female. "Whether there is any more fundamental similarity, other than their activity, between the male gamete of an Alga and a mammalian spermatozoa, remains rather a debatable question. The metabohsm of sperm is being very actively studied at the present time, both for its own intrinsic interest, and on account of its importance for the technique of artificial insemination. Reviews of recent work on mammalian semen and sea-urchin sperm will be found in Mann (1949, 1954) and Rothschild (195 id). 2. Oogenesis The formation of the egg-cell is a more comphcated and more lengthy process than the formation of the sperm. As a bearer of a haploid nucleus, the ovum has a somewhat simpler task than the sperm, since it does not need to produce any means of locomotion. But this simplification is more than outweighed by the fact that it is out of the cytoplasm of the ovum that the main structures of the embryo must be formed. The egg must 32 PRINCIPLES OF EMBRYOLOGY in the first place contain sufficient reserves of nutriment to keep the young animal aHve till it can obtain its own food, and although some of these stores can, as we have mentioned, be provided outside the egg-cell proper, yet in most cases such an expedient is only resorted to after the ovum Figure 2.2 Structure of a typical mammalian spermatozoon (that of the ram). On the left a light microscope picture of a complete sperm, on the right an interpre- tation of the structure of the middle piece as revealed in the electron micro- scope. (From Randall and Friedlaender 1950.) itself has been loaded to capacity. Further, apart from reserves of food, the egg cytoplasm must embody in some way the structural basis out of which the embryonic body can be formed; and this, one can see, must involve an elaborate process of preparation. It is not surprising, therefore, THE GAMETES 33 that the comparatively simple maturation of the sperm is profoundly modified in the differentiation of eggs (Fig. 2.1). The essential feature of the modification is the intercalation of a long stage of growth into the sequence. This occurs, in oocytes, before the first maturation division is completed; usually, in fact, the first division starts, and then goes as it were into a state of suspended animation while the cytoplasm and even the nucleus enlarge to many times their original volume. In some eggs (most invertebrates), the maturation divisions are not resumed until stimulated to do so by fertilisation. In others, such as most vertebrates, the egg completes its first division, but sticks again in the middle of the second until fertilised; it is only in a few types (e.g. echinoderms) that both the maturation divisions are completed before the eggs are shed from the ovary. The intercalated growth stage not only interrupts the continuity of the maturation divisions, but also modifies the relation between the division of the nucleus and the cytoplasm. It is as if the production of one full-sized egg were as much as one oocyte can manage; if, in the middle of its process of growth, each oocyte were to divide into two and then again into four equal parts, it would have to produce enough substance to make four eggs. The necessity for such an enormous achievement is avoided by making the cytoplasmic divisions extremely asymmetrical, only a tiny lump of cytoplasm being cut off from the main body of the egg, which remains substantially intact. The first of the lumps is formed, in marine eggs, at the end of the egg which floats uppermost. This end is known as the 'animal pole' (the opposite, heavier end being the vegetative pole'). The small cell produced by the first maturation division is thus known as the first 'polar body'. In the second maturation division it may divide again, while a second similar small body is given off from the egg. Thus the maturation divisions, instead of producing four ova from one oocyte, finally give rise to one ovum and three polar bodies. The latter soon degenerate, and, except in very pecuHar circumstances, play no part in the development of the embryo. During the growth of the oocyte, there is considerable activity both in the nucleus and in the cytoplasm. The former enlarges greatly, becom- ing a so-called 'germinal vesicle', filled with a voluminous nuclear sap. It is one of the great gaps in our knowledge of oogenesis that we know so little about the constitution of this sap, except that it is rich in sulphydryl- containing proteins (Brachet 1952^; Brown, Callan and Leaf 1950)- The chromosomes, arrested at some stage in meiotic prophase, usually tend to enlarge and become less densely staining. In highly yolky eggs which have a long growing period, this expansion of the chromosomes 34 PRINCIPLES OF EMBRYOLOGY proceeds very far, with the production of pecuhar so-called 'lampbrush' forms in which the basic chromosome threads (the chromonemata) are clothed in a fluffy mass of thin hair-like projections (Fig. 2.3). Chromo- somes at this stage stain very weakly in many of the dyes for which more normal ones show great affinity, and in particular they are difficult to stain in the Feulgen reagent which is more or less specific for desoxy- ribose-nucleic acid; nevertheless it appears that they never entirely lose r\ Figure 2.3 Structure of the loops of 'lampbrush' chromosomes from the germinal vesicle of the newt oocyte. The chromonema runs horizontally across the drawing. It bears small swelhngs (chromomeres) which are double. From these arise loops, which are usually symmetrical about the axis of the chromonema, but asymmetrical along the length of the chromonema. (From Callan, unpublished.) their stainability in this dye, and it is probable that desoxyribose-nucleic acid, which in all other circumstances appears to be an essential constituent of chromosomes, is present on them throughout oogenesis also (Callan 1952). Accompanying the expansion of the chromosomes, there is also an enlargement of the nucleolus, which often throws off smaller bodies so that the nucleus comes to contain many nucleoli (Fig. 2.4). The chemical con- stitution of these is different from that of the chromosomes; they contain much ribose-nucleic acid but no desoxyribose, and also much protein of a basic type involving arginine. Substances of the same general kind as THE GAMETES 35 those present in the nucleoH can be found in the cytoplasm, particularly in the immediate neighbourhood of the nuclear membrane, and it has been suggested that the nucleoli are important sites for the synthesis of basic protein-nucleates, which pass through the nuclear membrane and later control the synthesis of further cytoplasmic proteins (p. 382). The extru- sion of quite large droplets of nucleolar material into the cytoplasm can Figure 2.4 An early and a middle stage in the development of the germitaal vesicle in the oocyte of the snail Helix aspersa. The nucleus contains a large nucleolus, and by the later stage shovioi there are many small nucleoli in addition. The dotted region in the cytoplasm is the 'yolk nucleus'. (From Serra and Lopes I945-) be clearly seen in some forms (Fig. 2.5, p. 38). It is probable, indeed, that ribose-nucleic acid compounds are some of the most important consti- tuents in the cytoplasm of the growing oocyte, since they seem to be involved in the protein synthesis which must be taking place very actively there. During the grov^h of the oocyte they increase in amount, but not as fast as the total volume of the cell (Osawa and Hayashi 1953). Caspers- son and Schultz (1938) claimed that in Drosophila the quantity of such nucleotides in the oocyte was increased in eggs formed in mothers which 36 PRINCIPLES OF EMBRYOLOGY have extra heterochromatin (in the form of a supernumerary Y chromo- some) but this has been disputed by Callan (1948), and it is not clear what relation exists, if any, between the cytoplasmic and nucleolar ribonucleic acid on the one hand and the heterochromatic parts of the chromosomes on the other. In spite of the ribonucleic acid materials apparently given off from the nucleus, it is probable that the latter is not the main site of synthesis in the oocyte, hi fact, Brachet (1952I)) has argued that the nucleus contains less than its due share of the cellular enzymes, and, for instance, accounts for only a very small fraction of the respiration of the cell; this result, however, was probably a consequence of inadequate methods of cultiva- ting the isolated amphibian germinal vesicle with which he worked, and as far as respiration is concerned, the nucleus is probably just as active as the cytoplasm (Callan 1952). It is in the latter, however, that the major synthesis of the food reserves takes place. In many organisms there appears in the cytoplasm of the young oocyte a body which has received the name of the 'yolk nucleus'. It is particularly well seen in the eggs of some spiders, in which it has a laminated structure and is birefringent. In Amphibia it is represented in the young oocyte by a small mass in the cytoplasm which later expands and disperses to form a peripheral sheet lying just below the outer surface of the cell. It is usually considered to be constituted of mitochondria, and histochemical tests reveal the presence in it of fats, including the pecuhar phosphatides which give the so-called plasmal reaction, and of several enzymes such as indophenoloxidase, dipeptidase, etc. It appears almost certain that the yolk nucleus is the seat of a particularly active synthesis of fats and proteins. The reserve foodstuffs in eggs are often collectively referred to as 'yolk'. But this is really a loose use of the word. Strictly speaking, the true yolk is only one part of the reserve; it consists of platelets or lumps of a protein- lipoid substance. Besides it, the reserve contains globules of more or less liquid fat, and granules of carbohydrate, which are usually in the form of glycogen. In ova which contain fairly small quantities of reserves, these materials may be scattered more or less evenly throughout the cytoplasm ; such eggs are known as 'ohgolecithal', meaning that they contain little yolk. In most eggs with more than a very small quantity of reserve material, this is accumulated towards one end, the heavy vegetal pole referred to above; such eggs are called 'telolecithal'. There is a whole range of them, from only moderately yolky forms to bird or teleost eggs, in which the enormous mass of reserve food almost swamps the tiny patch of living cytoplasm. As a very rough general rule, the more highly THE GAMETES 37 evolved animals have more yolky eggs. Thus many marine invertebrates have rather little reserve, since their embryos can at a very early stage obtain nutriment from the microscopic living creatures of the sea. Verte- brates, in which the embryo cannot feed itself until it has developed a mouth and a gut, have much more yolk; even the amphibian egg is packed with yolk platelets, most fish have still more, and reptiles and birds most of all, as well as extra-ovular reserves in the form of the 'white'. But the rule breaks down for mammals, in which the embryo is fed through the maternal placenta; although the monotremes, the most primitive representatives of the mammal stock, have eggs nearly as yolky as reptiles, in the true mammals the ovum contains scarcely any reserve. Most insect eggs, on the other hand, have a great deal of yolk, which is accumulated towards the middle instead of at one end (these are spoken of as centrolecithal eggs). 3. Follicles and membranes During growth within the ovary, the cortex of the egg is usually closely invested by a layer of so-called 'follicle cells' which, presumably, play a major part in transmitting the materials for the growing oocyte; they may also be the main determinants of the cortical structure, although this is not definitely known. In mammals, the layer of follicle cells becomes very tliick ; in fact they increase to a largish spherical mass, within which a secretion is formed which hollows out the mass until the oocyte is hanging from a sort of stalk. This secretion contains the 'follicular hor- mone' which produces oestrus in the female mammal. When the egg is ripe, the follicle bursts and the egg, still surrounded by a layer of follicle cells, is set free to reach the Fallopian tubes and thus travel down to the uterus ; meanwhile the remains of the follicle forms the 'yellow body' or corpus luteum, from which is secreted the luteal hormone, an important factor in pregnancy. In other animals, the follicle cells are less in evidence, although probably always present. In insects the eggs are arranged in strings in the ovary; and there may be no special nutritive cells, or a group between each egg, or a single group at the end of each string wdth projecting strands leading down to the growing eggs (Fig. 2.5). The follicle cells or 'nurse' cells, as they are also called, are themselves often the site of active synthesis. In Drosophila and other insects their nuclei are polyploid, the chromo- somes having divided frequently without any accompanying division of the cytoplasm (Painter and Reindorp 1939) ; this phenomenon is often found in secretory or synthetic cells in insects. In most cases the sub- stances formed in the nurse-cells are passed almost completely into the 38 PRINCIPLES OF EMBRYOLOGY oocyte, so that the nurse-cells have almost withered away by the time the oocyte is fully grown. The processes of oogenesis often include the production of special pro- tective membranes to clothe the egg-cell, though a few types of marine Figure 2.5 Figures a, h, c show various types of 'egg-strings' from the ovaries of insects. In a the eggs are in simple foUicles (e.g. Orthoptera); in h the foUicles are accompanied by nests of larger 'nurse cells' (e.g. Coleoptera, Drosophila); in c there is a larger group of nutritive cells at the end of the string, with chan- nels leading to each egg (e.g. Hemiptera). (After Korschelt and Heider.) Figure d shows the discharge of material from the nucleolus of the germinal vesicle mto the oocyte cytoplasm in Limnea. (From Bretschneider and Raven 1951.) eggs are shed completely naked. Egg membranes are of three kinds; those which are strictly part of the ovum itself, being secreted by its outer surface; those which are formed by folhcle cells; and those which may be laid down by the oviduct during the passage of the egg away from the ovary. The first kind are usually known as the vitelhne membrane, the second as the chorion, while the third may have a variety of names such THE GAMETES 39 as egg capsule, or shell. Vitelline membranes are of very general occur- rence, except in the few naked types of eggs. Chorions are not found quite so often; the insect egg provides a good example of them. The tertiary membranes are particularly well developed in many vertebrates (e.g. the shell and albumen of the bird's egg) but occur also in many other classes (e.g. the egg capsules of molluscs). In some animals, the membranes may be formed before fertilisation, and it is then common to fmd that a special opening (known as the micropyle) is provided, which allows for the passage of the spermatozoon. The evolution of suitable egg membranes, and of eggs able to develop inside membranes, was one of the most crucial steps which had to be taken by animals in the colonisation of dry land (cf. Needham, 193 1). All animals provide enough organic food reserves to keep their embryos ahve until they can feed for themselves, but marine invertebrate eggs do not contain enough of either water or salts; these are absorbed by the embryo from the sea. The necessity for such salts has probably been a handicap to invertebrates in the colonisation of freshwater, and explains the relative poverty of freshwater as compared with marine invertebrate faunas. Fish go one better and provide all the salts their embryos will need, but they also do not cater for the water requirements. The same is true of most Amphibia, though in a few enough water is present in the egg to make possible a very much speeded-up development in a damp spot (e.g. the tree-frog Hyla). By this stage in evolution, eggs had become quite large, so as to contain enough organic matter for the development of an embryo which becomes rather compHcated before being able to feed. The reptiles began the next stage, that of enclosing the egg in a shell which could include and retain suffcient water to last till feeding can start, hi many of the turtles, the process is incomplete, and some moisture has to be absorbed from the wet sand in which the eggs are deposited, hi birds the problem has been fully solved; and in mammals it is, of course, circum- vented by the device of uterine development. It is interesting to note that water birds, instead of taking advantage of their environment to relax the effort to retain water in the egg, still provide sufficient stores for the developing embryo, and even evolve a waterproof shell to prevent more water entering. This is an example of a rather general rule of evolution, often known as Dollo's law, which states that evolutionary changes are irreversible. If a later animal returns to a set of circumstances similar to those in which one of its remote ancestors lived, it nearly always meets them, not by an exact return to the ancestral adaptation, but by some new expedient. The evolution of an egg-shell which would retain water solved one of 40 PRINCIPLES OF EMBRYOLOGY the problems of land life only to raise others. The shell which encloses the water also keeps in the nitrogenous waste products of embryonic meta- bolism. It is a rather general rule that during the earhest stages of embryonic life the main reserve foodstuff utihsed is carbohydrate (glycogen) ; next comes a stage when the protein is consumed, and last of all the fat (but see p. 454). It is the second of these, in particular, which gives rise to large amounts of nitrogenous waste. Animals with shelled eggs cannot avoid using such materials altogether, although they contrive to become very efficient in converting yolk protein into body protein without producing much waste; and they manage also to start rather early to consume fat, which has the added advantage that it produces some extra water as a fmal product of its oxidation. But even so, there is a good deal of nitro- genous waste to get rid of In the simplest marine forms, this is excreted as ammonia, a highly poisonous but rapidly diffusible substance. In fish, more efficient excretory organs are produced, and the waste products are got rid of in the less poisonous, but less diffusible form of urea. It is characteristic of land animals, that most of their nitrogenous waste is excreted as uric acid, which is a very insoluble substance. The reason why this mechanism has been evolved is almost certainly in order to cope with the situation of the embryo within its water-retaining shell; being unable to get rid of its waste products it needs to deposit them in a form which will stay put. Thus the exigencies of ovular life have a lasting effect on the metabolism of the higher vertebrates, though mammals, which escape the closed box of an egg-shell, have returned to urea as their main excretory end-product (a fact which does not fit very well with Dollo's law mentioned above). It is worth remarking that in animals which eventually excrete uric acid, we find that there are short early stages which excrete ammonia, like the earliest ancestors, and urea, like the rather more recent ones (Needham 193 1, 1942). This is a biochemical example of the phenomenon of 'recapitulation', which we have discussed as it apphes to morphological events (p. 9). 4. The morphogenetic structure of the egg The nutritive materials in the ovum are more or less 'inert' in that they play little part in determining the structure of the developing embryo. As we have seen, the yolk usually occupies a definite position within the egg-cell, but it can be shifted about, for instance by centrifuging the egg, without making any great difference to the course of development. The underlying basis of the embryonic body is to be found rather in the non- yolky cytoplasm. This is often optically clear and fairly homogeneous, THE GAMETES 41 and is therefore rather difficult to investigate. Many eggs contain, besides food reserves, a number of other granules, which may be grains of pig- ment, or mitochondria at which enzyme activity occurs, or may be of a so-far-undetermined nature (Fig. 2.6). We shall have to describe later eggs in which different regions of cytoplasm can be recognised by the different types of granule which they normally contain (e.g. in ascidians p. 106). But again, at least in many cases, the larger granules do not themselves deter- mine the development of the different regions, since, like the yolk, they can be shifted by centrifuging without affecting it. On the other hand. A. Genetic bast's: I Chromosomes 'iTh/monucttlc icid Figure 2.6 The internal constituents of the oocyte of the snail Limnea, as seen after centrifugation. On the right the distribution of the various components is shown diagrammatically, on the left is a drawing of an actual section. (After Raven 1948, and Bretschneider and Raven 1951-) we shall find examples (again for instance in ascidians) where, if the centri- fuging moves the actual clear ground substance of the cytoplasm, togedier with the ukra-microscopic granules, the ensuing development is pro- foundly altered. In such cases, it is clear that the different parts of the egg cytoplasm are endowed with different developmental properties. In other eggs, such as those of echinoderms, one can move even the internal cytoplasm around a good deal without interfering with develop- ment. But even in such cases, there are probably always some structures in the egg whose integrity is essential for the formation of a normal embryo. The most frequent location of this essential structure is the surface layer. The cytoplasm here is generally stiffer and more elastic than 42 PRINCIPLES OF EMBRYOLOGY It IS Within the egg-cell, forming an external layer known as the ecto- plasm or cortex. It may be entirely clear, or it may contain a special layer of granules; and it may have a definite external pelHcle or viteUine membrane-m any case, we shall see that a new membrane frequently forms from it at fertilisation. The cortex is so stiff that it is difficult to move by centrifuging, and, in eggs where normal development occurs after rearrangement of the interior, it seems that the reason is that the essential normal structure is still retained by this elastic external layer We know very little about the nature of this essential structure. It is often spoken of as a 'gradient'; but this means little more than that the animal end of the egg differs from the vegetative and that there is a gradual transition between them. Probably the essential properties of the differ- ent regions depend on sub-microscopic structures in the protein frame- work of the cortical cytoplasm; but if this is so, we shall have to await the development of new techniques of investigation before we can learn much about them (Fig. 2.7). 'MmiP Figure 2.7 Three fundamentally different types of egg structure. On the left, a 'mosaic' egg with locahsed regions of cytoplasm (the oligochaete, Tnhifex, after Penners). Centre the echinoderm egg, with a general polarity, indicated by the dark and light circles, and two opposing gradients, shown by the plus and minus signs (after Harrison). Right, an amphibian egg, with a vegeta- tive-anmia gradient dependent on yolk concentration! and a cortical gradient falling off from the position of the future blastopore. (After Dalcq and Pasteels.) In spite of the fact that our knowledge of these structural properties of the egg-cell is so slender, one must not overlook their fundamental im- portance. When we discuss the eggs of the different kinds of animals, we shall m every case fmd that the eventual origin from which the whole later development sprmgs is the orderly arrangement of essential parts of the ovum. We must therefore enquire a little more deeply how this THE GAMETES 43 arrangement is brought about. In particular, what is the relation between it and the hereditary factors or genes which determine the detailed charac- ter of the adult organism ? It was for a long time argued by many biolo- gists that genes affect only minor and trivial processes in the latter stages of development, while the major outlines of the animal body are deter- mined not by them, but by the nature and structure of the egg cytoplasm. This theory made no suggestion as to what determined the nature of the egg in its turn, and was therefore always somewhat incomplete. But this was not its worst fault; in the only suitable cases which have been pro- perly investigated, we have evidence that the theory is incorrect. There are not many examples of animals in which there are two or more variants in the basic structure of the egg cytoplasm, but a few cases are knoMOi. For instance, the eggs of molluscs cleave with a spiral pattern (p. 60) ; and this spiral may be either right- or left-handed, a few species having variants of the two kinds. In the pond-snail Limnea, the inheritance of such variations was studied by Boycott and Diver (1923, 1930). The right-handedness or left-handedness of an egg depended in a straight- forward way on the genes in the mother in whose ovary it was formed. This is a clear case of a difference in egg cytoplasm, which is inherited by means of genes, exactly as are differences, for instance, in the formation of pigment in an eye-cell. The only slightly odd feature of the situation is that, whereas one normally looks for pigment within the cell in question, we fmd it more convenient to diagnose the nature o£ Limnea egg cyto- plasm by waiting to see which way the fertihsed egg will cleave; but that is a mere matter of the technique of study. The important point is that in this case we can prove that the fundamental outline of the embry- onic organism is based on the egg cytoplasm, but that this in its turn is determined by genes, just as any other character is (Fig. 2.8). It is still quite uncertain how these genes operate. One possibility is that they influence some asymmetry which might exist in the protein molecules of the egg cytoplasm ; but this is rendered unlikely, though not perhaps impossible, by the fact that the structure of the sperm, which are also spirahsed, is not affected by the genes which control the symmetry of the eggs (Selman andWaddington 1952). Other examples of the control of the egg cytoplasm by the genes in the maternal ovary are provided by the 'female-steriles' in Drosophila (p. 135). We may almost certainly conclude that in all eggs the basic structure of the cytoplasm is laid down in the maternal ovary while the ovum is being formed, and that it is as fully dependent on the genes of the mother as is the character of her eyes or skin or hair. If this is so, we should expect to fmd a regular relationship between the main structure of the egg and 44 PRINCIPLES OF EMBRYOLOGY the position in which it lies in the ovary. This question has not had as much attention in recent times as it deserves. It is known that in many insects with elongated, banana-shaped eggs, the long axis of the egg always lies along the long axis of the insect; in some invertebrates, the position of the egg nucleus and of the accumulation of yolk has a definite relation to the blood supply, and the same has been claimed to be true of frogs eggs. But in most of the types of eggs in which the internal structure is clearest (e.g. ascidians) we know very little about the geometry of their formation within the ovary. Figure 2.8 The 4-cell stage and the adult shell of dextral {1, 2) and sinistral (3, 4) Linmea peregra. (After Robertson 1953.) (s) Shows part of the tail o£Limnea sperm consisting of three large and one small strand wound, always in a dextral spiral, round a central core. (After Sehnan and Waddington 1952.) SUGGESTED READING Rothschild 195 id^^ .. -;li J> f^ \K W^i FiGUKE 4.5 Maturation and first division of the egg of Tubifex, seen from the animal pole ; a, soon after laying ; b, formation of protuberances during the extru- sion of the first polar body ; c, between first and second polar body divisions ; d, extrusion of the second polar body ; e, before first cleavage of fertiUsed egg (the animal pole plasm shaded) ; /, early stage of cleavage, pole plasm elongated;^, h, stages of cleavage showing protuberances; i, two cell stage (the AB blastomere above, CD below). (After Woker 1944.) produced by an interaction between the cleavage spindle and the cortex in its neighbourhood, which has a structure which differs in the different parts of the egg. Lehmann claims that this structure is to some extent visible in the living egg, which shows a pattern of nine to fifteen sub- cortical meridianal thickened strands of the heavy fibrillar type of cyto- plasm which he names 'plastin'. This material is, however, at least to some extent, shifted by the centrifugation, and the structure which persists in the cortex of the centrifugal eggs is perhaps not due to the plastin, but to some associated structure in the cortex itself. 70 PRINCIPLES OF EMBRYOLOGY There is normally also an accumulation of plastin around the nucleus. With inild centrifugation in a polar direction at a stage shortly after fertihs- ation, all the plastin is driven to the centrifugal end while the nucleus still remains near the centripetal end, surrounded only by yolky cytoplasm. In such eggs the nucleus shows no sign of entering into division, which presumably indicates that the nuclear processes are normally initiated by a reaction which involves the surrounding plastin material. Further, no deformations of the cell cortex occur, which again demonstrates that they are initiated by the nuclear division process. These interactions be- tween cortex and nucleus in Tubifex are somewhat reminiscent of those between the nucleus and the cortical granules shortly after fertihsation in the echinoderms, as described by Allen (p. 51). It appears, then, that the pattern of cleavage is determined by inter- actions between the spindle and the cortex. We have now to consider the nature of the forces which bring about the deformation of the cell and its division into two. There are a number of theories in the field. As the first group we may take those which suppose that the nuclei and spindle apparatus continue to play in important part throughout the whole course of the division. For instance, Gray (Review: 193 1), from studies on the sea-urcliin egg, suggested that the asters continue to grow until all the available cytoplasm is incorporated into two spheres of radially arranged material at the poles of the spindle, and that the cortex passively accommodates itself to these two masses, which form the two first blastomeres. Dan (Review: 1948), working on the same form, suggests that the astral rays are actually attached to the cortex, and he shows by means of a model how, if this were so, a contraction of the rays would cause the bending in of the cortex and at least the beginning of the process of division. However, these mechanisms certainly do not operate in all types of eggs, if indeed in any. For instance, cleavage in the amphibian egg can continue quite satisfactorily when a large amount of the internal contents has been removed so that the cortex is quite flaccid and the asters imable to produce any internal turgor. Moreover, after a cleavage furrow has begun to form it can extend over a region of cortex which has been isolated from the spindle by the insertion of a strip of cellophane, which must certainly prevent the attachment of any astral rays (Waddington 1952^; Mitchison 1953 has similar evidence in echinoderms). (Fig, 4,6.) Finally Swann and Mitchison (1953) fmd that if sea-urchin eggs are treated with colchicine at anaphase, when the chromosomes have separated but the division of the cell body has not yet begun, the asters disappear but the cleavage occurs normally. Thus it seems fairly certain that although the division spindle initiates the process of cleavage it does not play any straight- CLEAVAGE Jl forward mechanical part in carrying the process through to completion. It seems that the main active agent must be the cortex itself. Probably the most widely accepted theory is one which ascribes the cleaving of the cell to the contraction of a ring of cortex around the posi- tion of the future furrow. This theory, which has recently been defended by W. H. Lewis (195 1) and Marsland (195 1, Marsland and Landau 1954) is the one which suggests itself most naturally when one looks at dividing 5-2.5 5-42 5.50 Figure 4.6 The independence of the cleavage furrow, once it has started, of contact with the spindle. In a newt's egg which was just starting to cleave at 3.23 p.m., a strip of cellophane was inserted under a region through which the furrow should extend. Within the next half-hour it did actually extend through the area. (From Waddington igs^d) cells and tries to think how their behaviour might be explained in terms of cortical activity. There is, however, rather little direct evidence for it. And, as Mitchison (1952) points out, there are some difficulties in it when one examines the matter more closely. In the first place, if the cell is to be constricted completely into two parts, the ring of contracting material would have to contract away to nothing. This, Mitchison argues, would seem to be an unlikely event, if one thinks of it in terms of a contraction like that of muscle: it is perhaps not so unplausible if one pictures the contracting ring as similar to the ectoplasm at the posterior end of an advancing amoeba, which liquefies after it has finished contracting. Again, 72 PRINCIPLES OF EMBRYOLOGY when a cell divides its surface area must increase; in the case of a spherical egg becoming converted into tv^o spheres, the increase is about 26 per cent. A contraction of the ring of the furrow would therefore have to be compensated by a large increase in area elsewhere. The main evidence on which Marsland rehes, for support of the 'contracting ring' hypothesis, is the demonstration that if gelation of the cortex is prevented (by high hydrostatic pressure or low temperature) the development of the cleavage furrow is inhibited. This might be due, as he suggests, to an abolition of the contraction; but it might equally be a consequence of the failure of the ungelated cortex to transmit the stress from the expanding regions to the furrow in the way required in the mechanism postulated by Mitchison and Swarm. Thus the evidence is not fully conclusive. Mitchison and Swann (1955, see Mitchison 1952) have recently turned the conventional theory upside down and suggested that the prime mover in cell cleavage is not a contraction of the furrow region but an expansion of the other parts of the cortex. They suppose that the cleavage process is initiated by substances released by the two separating groups of chromosomes (there seems no reason why one should not attribute the activity to the centromeres, rather than to the chromosomes themselves, in accordance with the points made on p. 66), This substance would diffuse out into a more or less dumbbell-shaped region which, in a spherical egg, would reach the cortex first at the equator opposite the poles of the spindle (Fig. 4.7). The substance is supposed to cause an expansion of the cortex, and this would begin in the same region. Meanwhile the ring of cortex which hes in the plane of the equator of the spindle (and therefore in- FiGUM 4.7 Diagram of cleavage in die sea-urchin. (After Mitchison 1952.) CLEAVAGE 73 eludes the poles of the egg) will become flattened for two difierent reasons. Firstly, Mitchison claims that there seems always to be some tension in the egg cortex, so that when it expands in one place, the other regions will tend to contract; and secondly, the expansion of the cortex on the egg equator is supposed actually to push the rest of it towards the plane of the spindle equator^. These two processes will, in fact, cause the future furrow not merely to flatten but to bend inwards. This will bring it in contact with the expanding dumbbell-shaped area containing the diffusing active substance, and when this happens the cortex in the furrow will be acted on by the substance and caused to expand; and this expansion com- pletes the formation of the cleavage plane. This theory was worked out in the first place from studies on echino- derm eggs. In that form there is a fair amount of support for various parts of it. Thus Mitchison and Swann have presented evidence that the cortical changes leading to cleavage begin in the equatorial regions of the egg, opposite the poles of the spindle, in the form of a decrease of the birefringence and hght-scattering properties of the cortical material. Although the structure of the cortex is not at all well understood, it is reasonable to suppose, as IVlitchison suggests, that it is made up largely of protein chains folded at right-angles to the surface, and that the changes in birefringence are connected with an expansion in area. Again, Dan and Ono (1954, see Dan 1948, 1954) have followed the movements of small particles of kaolin attached to the cell surface. They found that there was an expansion starting opposite the poles of the spindle and spreading towards the future furrow. The furrow region itself at first contracts somewhat, and then expands greatly as the new plane of division cuts down into the depth of the cell. Finally, Mitchison and Swann (1955) have devised an apparatus for measuring the deformabihty of the cell surface. The tip of a small pipette is brought against it and a negative pressure or suction appHed; one can then measure the height of the small protuberance which the suction raises on the surface. With this apparatus they showed that shortly before division the cortex becomes much less easy to deform. They suggest that this is not due to an increase in a tension (such as a surface tension) in the cortex, but rather to that layer becoming stiffer or less plastic. They argue that this makes it easier to suppose that the expanding regions opposite the poles of the spindle can successfully push the rest of the cortex into the furrow region. ^ The student is warned to beware of the verbal pitfalls that can occur from the fact that the long axis of the first cleavage spindle is perpendicular to the animal-vegetative axis of the egg. 74 PRINCIPLES OF EMBRYOLOGY Raven (1948) has estimated the 'tension at the surface' of the eggs of Limnea from fertihsation till first cleavage. His method w^as to centrifuge the egg for 5 minutes at a moderate speed, which gave a force of i86og. Observation of the distinctness with which the internal contents become stratified gives an indication of the viscosity of the cytoplasm, while the extent of the elongation of the egg allows one to draw some tentative conclusions about the deformability of the surface. The elongation will, however, also be affected by the degree to which the internal con- stituents separate into defmite layers, so that the viscosity changes some- what obscure the picture of the alterations in the properties of the surface. Raven finds that the viscosity is low immediately before each of the first three cleavages, but rises as the furrow makes its appearance, and reaches a maximum about 10-15 minutes later. The data on the surface tension are not so clear cut, and only the first cleavage has been investi- gated. Raven finds that it is low immediately before the appearance of the furrow. His diagrams show, although he does not refer to the fact in discussing them, that it rises steeply during the cleavage. It is not quite clear whether this rise occurs at a slightly later period than the similar increase in stiffness of the cortex of the echinoderm egg, but, apart from possible minor changes in timing, the phenomena seem rather alike in the two groups. These results are good evidence that some, at least, of the processes envisaged in Swann and Mitchison's theory actually occur. Moreover there is plenty of other evidence that expansions of the cell membrane are often associated with cleavage. Some of the most extreme examples of this are seen in those spirally cleaving eggs in which a polar lobe is formed (Fig. 6.2). But the mere occurrence of an expansion does not suffice to show that it is the prime mover in bringing about a cleavage of the cell, and there is some reason to doubt whether it is more than one out of a number of factors which may play a part. For instance, we have seen that in Tubifex there are considerable cortical expansions which cause the egg to throw out protuberances at the times of polar-body division and at the first cleavage division. The second cleavage division occurs first in CD blastomere and only somewhat later in the AB one, and is not accompanied by such a pronounced cortical expansion as the earlier ones, though the CD cell does elongate considerably and increase its surface area to a fair extent just before the cleavage occurs. Huber (1947) has studied the action of two anti-mitotic substances, naphthoquinone and phenanthrenequinone. He finds that the former has a particularly strong effect on the expansions, tending to suppress the formation of protuberances in the first division and the stretcliing of the CD cell in the CLEAVAGE 75 second. The second substance, on the other hand, leaves these processes relatively unaffected or even exaggerates them, but the cells tend to fail actually to divide into two, so that one finds, for instance, an elongated but undivided CD cell (Fig. 4.8). Huber therefore concludes that cell division involves two different cortical movements, not only the expan- sion seen in the formation of protuberances, but also a contraction in the furrow region. A consideration of more yolky types of egg would also suggest that a mere expansion of the already existing cell cortex cannot be the only factor causing the cleavage. In the extremely yolky eggs of a sturgeon, FiGXJRE 4.8 The effect of quinones on cleavage : a, section through a 2-cell stage of the egg of Tuhifex; the nuclei are aheady in telophase of the second division and the CD cell (below) is markedly elongated preparatory to dividing in two ; b, a similar stage from an egg treated with 1,4-Naphthoquinone, note the slight elongation of the CD cell; c, after treatment with 9,10-Phenanthrene- quinone the elongation of the CD cell is exaggerated. (After Huber 1947.) for instance, the cleavage furrows eventually extend a long way from the position of the spindle, and it is difficult to see how the influence of a cortical expansion starting in the neighbourhood of the spindle poles could reach so far (cf Fig. 4.1). Moreover in such forms the second cleav- age furrow starts to form in the animal region while the first furrow is still working its way down towards the vegetative part of the egg. It seems unhkely that any system of expansions working through the cortex as a whole could control the furrows in such cases. It would be much easier to attribute the formation of the cleavage planes to factors located in the immediate neighbourhood of the furrows themselves. Studies on the moderately yolky eggs of the Amphibia suggest, indeed, that in them also cortical expansion is not such an important factor in cleavage as Mitchison and Swann suppose it to be in the smaller eggs of the echinoderms. Selman and Waddington (1955) have shown that there 76 PRINCIPLES OF EMBRYOLOGY is little movement of cortical pigment granules in the neighbourhood of the poles of the spindle and there is no noticeable cortical expansion there. However there is a considerable flow of the granules along the furrow as it first appears near the animal pole of the egg. This movement along the furrow would seem to indicate a contraction acting in this direction and at this place. Moreover if the eggs are viewed from the side it can be seen that just before division the whole cell heaps itself up so that its vertical height increases (Fig. 4.9). This movement is accompanied by an increase in the resistance of the cortex to deformation as measured by Figure 4.9 Side views of cleavage in a newt's egg removed from its vitelline membrane to show the 'rounding up' at the beginning of division. A, part-way through the first division; B, between first and second division; C, beginning of second division; D, second division completed. (After Selman and Wad- dington 1955.) the suction apparatus of Swann and Mitchison. This must involve a force acting in the cortex, which might be either analogous to a surface tension, or, more probably, due to an increase in the elastic constants of the mater- ial. In any case, the rising up of the egg suggests a decrease in surface area rather than an increase. It becomes rather unplausible, then, to attribute much importance to cortical expansion in this case; we seem rather to have to deal with a locaHsed contraction along the length of the developing furrow. There is, however, almost certainly another factor involved. When an amphibian egg cleaves, the surface between the two blastomeres is almost unpigmented, and thus differs sharply from the cortex of the CLEAVAGE 77 uncleaved egg. It seems that it must have arisen de novo. Schechtman (1937) suggested that it is formed by the growth of the cortex into the depth of the furrow. Mitchison and Swann argue that such growth takes place, not merely from the very edge of the original cortex, but by intus- susception of new material throughout the whole area of the infolding region; if this were so, the process becomes in effect a type of cortical expansion. A similar increase in surface area occurs in the furrow region of the echinoderm, but in this form it seems to take place after the cleavage plane has cut through the egg (Fig. 4.10). According to Dan (1954) \ .' <■• >CTV ra^^w xjf^iz • •« Figure 4.10 A. Section through part of a newt's egg at an early stage in the first cleavage. The future surface which will separate the two blastomeres is already indi- cated in the internal cytoplasm although there is not yet any furrowing of the external surface in this region. (After Selman and Waddington 1955.) J3. Three stages in the cleavage of an echinoderm egg. Small kaolin particles (indicated by short lines) have been placed on the surface. Note that they at first move into the furrow (2), but that later new cortex is produced in the depth of the furrow (dotted), so that the particles move out again. (From Dan 1948.) it also seems to be due to intussusceptive growth, accompanied by stretching. From this evidence, Mitchison and Swann conclude that the cortical expansion involved in their theory always includes an element of intussusceptive growth. But even if this is so, the amount of growth as compared with mere stretching must be so vastly greater in the furrow region than elsewhere as to amount to a qualitatively different type of 78 PRINCIPLES OF EMBRYOLOGY phenomenon. Moreover, Selman and Waddington find that sections of an amphibian egg in the process of division show that an indication of the future cleavage plane is developed in the cytoplasm over a much wider area than corresponds to the externally visible furrow. It seems very unlikely that the material making up this precursor of the division plane is directly derived from the original cortex. It looks rather as if it differen- tiates in situ (although it must be the cortex which initiates the process of differentiation). It gradually increases in extent, and also in thickness, and eventually spHts into two separate films which form the outer membranes of the two blastomeres in the region where they are in contact. If the vitelline membrane is removed, so that there is nothing to hold the two cells together, they tend to fall apart as they settle down on the bottom of the dish, and the white newly formed cell membrane is then extensively exposed in the depths of the furrow as one looks at the egg from the animal pole; it may be visible within the furrow even in eggs cleaving inside the membrane. The evidence available at the present time would seem, therefore, to suggest that several factors are operative in the process of cell cleavage, their relative importance differing in various groups of animals. These factors are: firstly, localised expansions of the cortex; secondly, an increase in stiffness of the cortex; thirdly, in the Ampliibia at least, an increase in the tangential force acting in the cortex; fourthly, a contraction localised in the neighbourhood of the furrow and directed along its length; fifthly, a formation of new cell membrane from the sub-cortical cytoplasm. It is possible that Swann and Mitchison are correct in ascribing the major importance to the first two of these in the echinoderm egg but it seems likely that in other eggs, particularly those with large quantities of yolk, the other three factors have to be taken into account. SUGGESTED READING Fankhauser 1948, Mitchison 1952, Swann 1952, Lehmann 1946, CHAPTER V ECHINODERMS I. Normal development The early stages in the development of echinoderm eggs are very simple, and provide classical examples of the two fundamental forms, the blastula and gastrula. Moreover, the physiology of these stages has been rather fully investigated. There is therefore much of interest in echino- derm development even for a general account of embryological principles, in spite of the fact that the later stages are highly complex. The simple, early stages of development lead to the formation, not of the adult, but of a larva, which is usually the so-called pluteus'. The insertion of a larval stage into the life-history is, of course, very common among invertebrates. Such larvae fulfd many functions; they may, for instance, faciUtate the dispersal of the species, as in many groups of parasites; or they may seem to have been evolved as the quickest way in which the egg can be con- verted into an animal capable of feeding itself The latter would appear to be the raison d'etre of the Pluteus; it is a httle animal which can swim by means of cilia, and feed itself from the minute life among the plankton. It is only after a considerable independent existence that it becomes con- verted, by a complicated metamorphosis, into the adult. In this discussion, we shall not attempt to deal with anytliing more than the first steps of development, by which the larva is produced (Reviews: Lehmann 1945, Horstadius 1939, 1949)- The cleavage of the echinoderm egg is total, and radially symmetrical. The first two cleavages are vertical, through the animal pole. The third is horizontal, running slightly above the equator, so that the upper four cells are rather smaller than the lower four. From this point onwards, the cleavages in the animal and vegetative halves take different courses. At the fourth cleavage, the animal cells divide into a flat ring of eight, while the vegetative ones cleave very unequally into two rings, four large 'macromeres' above and four tiny 'micromeres' below; the cleavage planes are thus nearly at right angles in the animal and vegetative halves. The same is true at the next cleavage, but here it is in the animal half that the division is horizontal, while in the vegetative half it is more or less vertical. At the sixth cleavage, all the division planes are horizontal, and thus we come to a stage with four rings of animal cells, two rings of macromeres, and two of micromeres, there being eight cells in each ring. 79 8o PRINCIPLES OF EMBRYOLOGY For our present discussion, it is not worth while tracing the cleavage in detail beyond this point (Fig. 5.1). Figure 5.1 The development of an echinoderm. Figure a-f show the early cleavages stages. The shading indicates the levels which have been used in grafting ECHINODERMS 8l During the early cleavages, a space is gradually formed between the cells in the centre of the mass. This enlarges as the cleavage progresses, and fairly soon after the last stage mentioned above, all the cells cohere to- gether to form a smooth hollow spherical ball with this space in the centre. This ball is the blastula, and the central space is the hlastocoel or blastula cavity. The simple spherical shape is the most 'typical' form the blastula can take; all the many variations which we shall find in other groups can be regarded as modifications of it. In the echinoderms there are few special features to notice; but one may remark that the surface of the blastula soon becomes ciliated, and the tiny embryo escapes from its membranes, and begins an independent Hfe soon after the cilia develop. After a short time, it grows a bunch of special long cilia (the apical tuft) at its animal pole. Soon after the apical tuft forms, the blastula loses its strictly spherical shape and begins to flatten at the vegetative pole, diametrically opposite the tuft. This is the first sign of gastrulation, which, in essentials, consists in the folding inwards of the vegetative part of the blastula to form a pocket pressed into the cavity of the blastocoel. This pocket is the first rudiment of the gut, and is usually known as the 'primitive gut' or archenteron. Its walls make up the endoderm, the innermost of the three fundamental layers out of which the embryo is built. The opening by which the gut communicates with the exterior is the blastopore. Mean- while, the rest of the surface of the blastula, which is not pressed inwards, forms the outermost layer or ectoderm. Between these, as we have seen, there should be a third or middle layer, the mesoderm. The main way in which the early development of the echinoderm differs from the general scheme which applies to vertebrates is that here the mesoderm originally lies, not between the ectoderm and endoderm, but right at the vegetative pole. It is formed from the micromeres and the material just above them. experiments; white is an-i, fine dots an-2, coarse dots veg-i, circles veg-2, and black the micromeres. Figure^ shows a section at the blastula stage, at which time ciha develop on the surface; h, beginning of gastrulation and formation of apical tuft of long ciha; /, the micromeres come free into the blastocoel to form the primary mesoderm p.tn.;j, appearance of primitive gut, with blastopore hi; from the tip of this secondary mesoderm [s.m) is given off, the general ciliation is not shown in this or later drawings ; k, hori- - zontal section through same stage as;; /, flattening of ventral side, the skeleton begins to be laid down by the primary mesoderm; »i, n, views of young pluteus larva from the original vegetative pole and from the side, showing the development of 'arms' from the comers of the ventral surface, and the junction of the tip of the primitive gut with the ventral ectoderm to form the mouth. The dotted hne in n shows how the original animal vegetative axis has become bent. (After Horstadius I939-) 82 PRINCIPLES OF EMBRYOLOGY The most vegetative cells begin to break loose separately into the blasto- coel even before there has been much infolding of endoderm. These are the 'primary mesoderm' cells, and they soon start secreting the fnst cal- careous spicules of the skeleton. A similar process of shedding cells into the blastocoel goes on particularly from the tip of the primitive gut as it folds in; this is the 'secondary mesoderm', which later forms the greater part of the skeleton and muscles. The pocket of endoderm pushed in to the blastocoel continues to elon- gate, producing a long fmger-shaped primitive gut. The process is know^n as 'invagination', a term v^hich is applied to all types of movement by which the endoderm and mesoderm are formed from the blastula; as we shall see, it covers several different sorts of foldings and cell migrations. In the echinoderms, it appears to be a simple in-pushing, like that pro- duced when one presses a thumb into a soft hollow ball. While this is going on, the first signs of bilateral symmetry appear. The embryo, now entitled to the name of gastrula, has become somewhat conical. It begins to flatten on one side, which is the future ventral side. The elongating primitive gut turns towards the upper end of this side, fuses with it, and eventually breaks through to form an opening. This is the rudiment of the mouth; and there is now a complete tube leading from it to the blastopore, which from now on functions as the anus. The gut soon begins to differ- entiate into an oesophagus, a stomach and an intestine; and meanwhile four long arms grow out from the comers of the flattened ventral side, while the opposite comer of the gastrula also elongates into a single thick spike. This completes the formation of the pluteus. 2. The gradient system Echinoderm eggs were some of the first with which experimentahsts tried to solve the fundamental physiological problems of development. They are easily obtained in large numbers; and it is simple to free them of their membranes (by squirting them through a narrow pipette, for example). Only their small size is an impediment; but this, so long a major limitation on the kinds of experiment possible, was largely mastered by the Swedish investigators Runnstrom, Horstadius and Lindahl, who combined subtle chemical methods of attack with the most dehcate manipulative skill. In the earliest experiments, which were mentioned on p. 62, Driesch showed that if the first two, or first four, blastomeres are separated by cutting along the cleavage planes, each isolated cell can form a complete well-proportioned pluteus. He concluded that these cleavages do not separate parts whose developmental fates have been already determined; ECHINODERMS 83 the later developmental history of a blastomere can be altered if the cell finds itself in an abnormal situation, as it does when isolated. This con- clusion still stands. The cleavage pattern can be altered by various treat- ments, and in spite of this, normal larvae are formed. Clearly the pattern of cleavage has no decisive effect on the pattern of develop- ment. Driesch went further in his conclusions and considered that every part of the early egg had the same potentialities. He argued that the formation of a complicated embryo from an egg all of whose parts were alike was an inconceivable achievement to be accomplished by a natural mechanism, and that its explanation demanded a non-natural agent, whose functions were to create order out of uniformity; this he called 'the entelechy'. But as a matter of fact, Driesch' s basic postulate, that all parts of the egg are similar, has turned out to be untrue; and the entelechy thereby loses its main support. The demonstration of differences within the egg has come from experi- ments in which the parts have been cut apart along horizonal planes. There is no need here to attempt to summarise the whole massive volume of evidence, and we will select one of the most demonstrative experi- ments— that in which the 32- or 64-cell stage has been dissected. Horstad- ius, who did the experiment, distinguished five zones. Anifiicil-i and animal-2 lie at the top, and below them are vegetative-i and vegetative-2 derived from the macromeres ; at the very bottom of the egg there are the micromeres. In the first part of the experiment, each of these zones was isolated. The essential feature of the result was that each zone devel- oped more or less in accordance with its normal fate within the embryo, only more so, if one may put it like that. For instance, an-i gave a pluteus which contained only the organs appropriate to the most animal region (e.g. apical tuft, no gut) ; but these organs were exaggerated, in the sense that the apical tuft spread out to cover nearly the whole surface. An-2 also gave larvae with no gut, and usually with apical tufts, which were often enlarged (although normally an-2 does not participate in the formation of the tuft, which is formed from an-i cells). Similarly the vegetative cells gave larvae containing vegetative organs, which again inight be exagger- ated. The micromeres unfortunately fail to develop anything when isol- lated, but veg-2 gives a larva with an exaggerated gut, which is often too large to fit inside the ectoderm and thus protrudes, the resulting embryo being known as an exogastrula. Veg-i is highly variable; sometimes it has exaggerated vegetative organs (e.g. gut), sometimes it gives a larva consisting mainly of animal organs; in a few cases it produces a reasonably normal pluteus. 84 PRINCIPLES OF EMBRYOLOGY B • • • • • •0« Figure 5.2 Combinations of the animal half with various levels along the animal- vegetative axis: A, the isolated animal half (an-i plus an-2) forms a blastula nearly completely covered with apical cilia, and later a larva with no gut; ECHINODERMS 85 Horstadius interpreted these results to mean that the egg contains two gradients, an animal one with its high point at the animal pole, and a vegetative one running in the opposite direction. Regions which are high on the animal gradient tend to form animal organs, and similarly for the vegetative. Moreover, Horstadius proceeded to show that the two gradients interact with one another. In a very beautiful series of experi- ments, he combined the various layers in abnormal combinations. He found, for example, that normal plutei were formed fairly frequently when an-i was combined with four micromeres, and that as the number of micromeres was reduced, so more and more 'animal' larvae appeared. An-2, on the odier hand, required only two micromeres to produce a normal larva, and became vegetative in character if more were added. Veg-i was usually swung over too much to the vegetative side by the addition of even one micromere; probably about a half would be enough to counteract its slight preponderance of animal tendencies (Cf. Fig. 5.2). Both in the experiments in wliich the various zones were isolated, and in those in which micromeres were grafted, there was considerable varia- tion in the resulting embryos. It appears that one is dealing not only with the interaction of animal and vegetative potentiaHties of various strengths, but that there is also another important factor, namely a tendency for the normal equihbrium between these two conditions to be restored, even when the two parts originally grafted together were not in perfect balance (see Lehmaim 1945, p. 64). We will fmd that a similar tendency to restore a normal equihbrium condition is a very frequent influence in most developmental events. It is an aspect o( individuation (p. 12). There are several other types of experiments in which individuation is strikingly exhibited in the echinoderm embryo. The vegetative region, and particularly the micromeres, have a very strong ability to influence their surroundings in such a way that the latter fit into the building-up of a complete or partially complete embryo. For instance, a meridional half may be combined with an animal half in the i6-cell stage (Fig. 5.3). From this a normal pluteus will develop, the part of the animal material lying next the micromeres being, as it were, absorbed into the develop- mental system of the latter and converted into gut. A rather similar result is obtained if a group of micromeres is inserted among the animal cells B, the addition of vcg-i restricts the size of the apical tuft, but again no gut develops; C, with veg-2 a relatively normal embryo is formed; D, with veg-i plus the micromeres, the embryo forms a gut which is abnormally large; E, the addition of the four micromeres to the animal half gives a near- ly normal larva, although their mass is much smaller than the veg-2 material added in C. (After Horstadius 1939.) 86 PRINCIPLES OF EMBRYOLOGY of an intact egg (Fig. 5.4). They continue developing in a vegetative v/ay, and, further, swing some of the neighbouring animal material into line with them, so that it too develops as gut. The animal gradient of the egg is exhibited by the fact that the nearer the animal pole the graft is placed, the smaller is the gut formed. It is important to note the similarities, and also the differences, between the production of a gut by the action of the grafted micromeres on the animal cells in the above experiments, and the 'embryonic induction' which is most strikingly seen in vertebrates. In both cases, a part of the embryo, when grafted into an abnormal situation, causes the material Figure 5.3 A meridional half of the i6-cell stage (uncoloured, on left, containing the whole an-veg axis) is combined with the animal half of the same stage (shaded). A normal embryo develops, shown in section on the right. (After Horstadius.) surrounding it to develop in a way which it would not normally have done. In true embryonic induction, however, the graft induces from its surroundings something of a different nature to itself; for instance, in the vertebrates, mesoderm can induce the formation of neural tissue, or in Linmea the gut can induce the formation of the shell-gland. In the echino- derms, the micromeres induce something which is either normally derived from micromeres or from the next most vegetative material; we are dealing with a tendency for the completion of the graft at the expense of its surroundings; moreover, these surroundings clearly play an important part in determining the nature of the structures induced. In the verte- brates, as we shall see, induction may sometimes show certain features which indicate a similar assimilative or individualising tendency, but it can also be a much more independent process, the inducer provoking the appearance of something which it does not in any way need in order to ECHINODERMS 87 complete itself. Thus vertebrate induction may involve both individua- tion and something else, which has been named 'evocation' ; in the early- stages of the echinoderm, we seem to have only the former. The hypothesis of two gradients fmds very strong support in the work described above. But innumerable questions immediately suggest them- selves. What are these things gradients of? Are they localised in any par- ticular part or structure of the egg ? And how do they produce their Figure 5.4 Grafts of the micromeres (black) at different levels along the an-veg axis. They migrate into the blastocoel cavity as primary mesoderm, and induce the formation of an archenteron from the material near them. This induced gut (indicated in black in the right-hand column) is larger the nearer the graft is to the vegetative pole. (After Horstadius.) effects ? None of these questions can be answered with any assurance, but knowledge is increasing rapidly about them. As regards the first, we know that the gradients can be influenced by chemical substances (Lin- dahl 1942, Gustafson 1950). The vegetative tendencies are strengthened by the action of lithium salts. If complete eggs are treated with lithium, they form too large a gut, producing exogastrulae similar to those formed when veg-2 is isolated. Similarly, isolated animal halves can be brought to develop into normal larvae by lithium. The animal tendencies are increased by early treatments with thiocyanide. This, and other similar 88 PRINCIPLES OF EMBRYOLOGY evidence, suggests that the gradients are fundamentally based on the rates of certain critical chemical reactions. Moreover, if young embryos are constricted, by being tied in a loop of hair, the degree to which the animal and vegetative gradients interact with one another depends on the width of the comiection between the two halves, which again suggests that diffusmg chemical substances are involved. We still have little idea what these substances are. Very careful measure- ments have failed to reveal any difference in respiration between the animal and vegetable halves. However, Child (1936) observed some years ago that by the blastula stage there is a double gradient in the rate of reduction of vital dyes such as Janus Green and methylene blue, which are indicators of redox-potential. There is a high level of reduction acti- vity at the vegetative pole, falling off towards the animal, and simul- taneously a weaker gradient running in the opposite direction from the animal pole towards the vegetative. Horstadius (1952) has recently studied these gradients in isolated animal and vegetative halves, and in animal halves into which micromeres have been implanted, and has shown that their behaviour parallels that of the postulated gradients of animal- vegetative tendencies. The biochemical meaning of the gradients in dye reduction is not yet understood, nor can one be certain whether they are related to the causes of the animal and vegetative gradients or are merely among their effects (Fig. 5.5). A perhaps more promising clue to the processes underlying the grad- ients is the fact that in rather late cleavage stages the vegetative region of the egg comes to require the presence of sulphate ions in the medium, and its development is inhibited in artificial seawaters from which they are absent. Lindahl (1942) suggests, on these and other grounds, that the reactions proceeding in the vegetative region give rise to aromatic waste products (formed from protein catabohsm) which are normally disposed of by being combined with sulphate. Horstadius and Gustafson (1954) have studied the animahsing and vegetativising effects of various amino- acids as well as substances which might be expected to play a role in carbohydrate metabolism. The latter group were, on the whole, animal- ising, which supports the suggestion that the animal tendencies are con- nected with carbohydrate metabolism; but the effect of the amino-acids, although usually towards vegetativisation, was not invariably so, and further work will be necessary before the experiments can be fully interpreted. Meanwhile there is another quite different theory in the field. Ranzi (195 1) presents evidence to show that lithium and thiocyanate have im- portant effects on the viscosity of solutions containing elongated protein ECHINODERMS 89 molecules, and he believes that their actions on the echinoderm egg are related rather to their influence on the physical condition of the cytoplasm than to any direct alteration of the chemical metabolism. Finally, a very unexpected point has recently been discovered by Lrndahl (1953) v^ho has claimed that, in the two species of sea-urchin he studied, the micromeres are haploid in chromosome number. It is not clear how this remarkable example of nuclear differentiation (cf. p. 3 54) is related to the high Vegetative' activity of these cells. As regards the localisation of the gradients, most authors agree that they must be in the cortex. The argument is primarily the negative one. Figure 5.5 Sections of early gastrulae stained with Janus Green, which is at first blue (large dots) changing to red (fine dots) as it becomes reduced; in the un- dotted areas the colour has faded altogether. A i, 2, 3, three stages over a period of 50 mins. in an isolated vegetal half, showing the reduction spread- ing from the most vegetative region. B i, 2, 3, a period of 55 mins. in an isolated animal half, the reduction starting in the most animal region. C, an animal half into which micromeres have been grafted, showing the induction of an archenteron, with an associated region of rapid reduction, and the slow progress of reduction in the neighbourhood of the apical tuft. D, micro- meres were implanted on the left between an-i and an-2 of a normal 32-cell stage, as in Figure 5.4 middle row; a region of rapid reduction has appeared in their neighbourhood. (From Horstadius 1952.) 90 PRINCIPLES OF EMBRYOLOGY that the internal regions of the egg can be shifted about by centrifugation without producing any profound result, so that the controlling gradients must be in the stiff ectoplasmic layer which is not displaced by centri- fugation. Finally, to the question of how the gradients work, we can offer at least the beginning of an answer. Although moderate centrifugation, sufficiently strong to cause considerable stratification of the cytoplasm, has no great effect on development, the situation is rather different follow- ing very intense centrifugation in a modem ultra-centrifuge. Using forces of the order of 45,000 g, on the unfertilised egg. Pease (1939) was able to throw down to the centripetal end a mass of small mitochondria-like granules. He found that if, owing to the orientation of the egg in centri- fuge, these had been collected at the animal pole, the vegetative pole which lacked them was unable to gastrulate; and so, mutatis mutandis, was the animal pole unable to develop if all the granules had been forced to the vegetative end. He suggested that these granules are the im- mediate agents of differentiation. At the present time, a great deal of attention is being paid to the be- haviour of various types of cell granules in developing echinoderms. They can be roughly classified into 'mitochondria', which are larger and become sedimented at relatively low speeds of the centrifuge (giving about 16,000 g), and 'microsomes', which are only sedimented at the highest speeds (giving about 100,000 g). The mitochondria can be detected by normal cytological techniques witliiti the cells of the embryo. It has been found (Gustafson and Lenique 1952, Lenique, Horstadius and Gustafson 1953) that in early stages they are distributed in a gradient, de- creasing in concentration from the animal to the vegetable pole (Fig. 5.6). In isolated animal or vegetative halves, and in halves which have been 'animalised' or 'vegetalised', the gradients become altered in a manner exactly parallel to that of the basic animal and vegetative gradients, or the gradients in dye reduction mentioned above. This makes it most probable, then, that the mitochondria are rather directly concerned with the fundamental developmental processes. Gustafson (1953, 1954) has made extensive studies on their metabohsm, and suggests that they are connected with the synthesis of the fibrous proteins of the apical tuft, which is the most characteristic animal organ. The mitochondria can be formed anew during development. Harvey (1946) centrifuged echinoderm eggs in a medium which had a gradient of specific gravity, and showed that under these circumstances, the egg may be split into several fragments, each containing the constituents of a particular density. Fragments made in this way may develop normally ECHINODERMS 91 even if they originally contain no mitochondria; but it is found that mitochondria gradually appear. It seems probable that they are formed from the cytoplasmic particles of smaller size (the microsomes). Hultin (1953^, b) has summarised a number of studies in wliich radioactive iso- topes have been used to follow the processes of protein synthesis in the cell as a whole and in the various groups of particles (see also Kavanau 1953). During the early cleavage stages, it is in the microsomes that this Figure 5.6 Graphs showing the relative density of the mitochondrial population (R.M.D., plotted vertically) at various levels of the animal-vegetative axis at 7, 14 and 26 hours of development (blastula to mid-gastrula). (From Gustafson 1954, after Lenicque, Horstadius and Gustafson 1953.) synthesis is proceeding most rapidly, but by the early gastrula the mito- chondria are becoming very active. Hultin suggests that the mitochondria are built up either from, or at least by the influence of, the microsomes. The reactions between these two types of particles are, however, probably reciprocal, since there is reason to believe that, once the mitochondria have been formed, they induce a high rate of activity in the microsomes, and that it is actually at these small particles that protein synthesis proceeds most rapidly. The evidence from work with isotopes suggests that this synthesis begins to get fully under way at about the time of gastrulation. 92 PRINCIPLES OF EMBRYOLOGY Perlmann (1953) has indeed been able to detect, immunologically, the appearance of at least one new antigenic substance (protein?) at that time. The study of the roles of the various types of cytoplasmic particle has only begun very recently and there is still much to do. Hultin emphasises a point v^hich is probably of considerable general importance, namely that we must think of the developing cell as containing populations of mitochondria and microsomes which are continually reacting on and influencing each other; and one may add, of course, that the nucleus with its contained genes must be also involved in the same general net- work of cause and effect. All the grafting experiments on the gradient system described above were carried out on the early cleavage stages, before the embryo has sixty-four cells. Isolations of animal and vegetative halves can be made at later stages, and it is found that the divergence of the isolate from its normal fate remains much the same until the early blastula stage (about ten hours after fertilisation). From that time onwards, the develop- mental fate of the regions rapidly becomes more fuced, so that, for instance, an animal half isolated from a later stage develops only the normal sized tuft of cilia, and a vegetative half only a normal gut. The developmental fate is not entirely determined in such halves, since if micromeres are implanted into animal halves isolated at various times, it is found that they can induce some vegetativisation even in halves which, if left isola- ted, would develop only into their original presumptive fate. The em- bryonic materials are, however, rapidly losing their lability, and by about sixteen hours after fertilisation, an animal half can no longer be affected by implanted micromeres. It is interesting to note that if an animal half is isolated at the 32-cell stage, and allowed to develop in isola- tion for some time before the micromeres are grafted into it, it loses its reactivity earlier than it would do as part of a whole egg ; presumably its isolation from its vegetative partner allows its animal tendencies to become fixed earlier. 3. The dorso-ventral axis It was stated earlier (p. 48) that the determination of a dorso-ventral axis, which confers a bilateral symmetry on the egg, is one of the funda- mental steps in development. It may well be asked how, and at what time, this step is taken in echinoderm development. As a matter of fact, the evidence suggests that there is some trace of bilaterality even in the unfertilised egg, presumably dependent on factors operating during the maturation of the egg in the ovary. If such eggs, or early cleavage stages, ECHINODERMS 93 are sectioned vertically, there are slight differences in development accord- ing to the plane of the section. These can be best interpreted by the hypo- thesis that one side already has a slight tendency to become ventral, and usually does so. If a ventral half is separated from a dorsal half, the ventral face appears in its original position in the former, but in tlie dorsal half, the axis may be reversed, so that the eventual ventral side of this half- embryo appears on the side which, in the undisturbed egg, would have been dorsal. This original organisation is only a labile one, and can be overcome by a number of different influences. It becomes determined at about eight hours after fertilisation. If eggs are strongly stretched before this, by being sucked into a tube with a narrow lumen, the dorso-ventral axis is altered so as to run along the length of the elongated egg. Strong staining of one end of such an egg with Nile Blue sulphate will cause this end to become dorsal; perhaps it would be better to say that it will cause the other end to become ventral, since it is probable that the ventral side plays the lead in the development of the axis, and that it is a suppressive action of over-staining which is the operative mechanism in the experiment. Many other chemical substances can influence the determination of the axis (see Review in Lehmann 1945). Gustafson and Savhagen (1949) have recently shown that weak solutions of detergents will suppress the develop- ment of the oral or ventral side entirely, so that radially symmetrical larvae are formed. A similar result was produced by Horstadius and Gustafson (1954) who treated the eggs shortly after fertilisation with antagonistic analogues (or 'anti-metabolites') of certain vitamins, etc. They suggested indeed that these substances were actually operating as detergents rather than by inhibiting the growth-promoting properties of the vitamins. SUGGESTED READING Horstadius 1939, 1949, Gustafson and Lenique 1952 or Gustafson 1953, I954, Hultin 1953^- CHAPTER VI SPIRALLY CLEAVING EGGS THE SPIRAL type of cleavage is sufficiently definite to allow one to recog- nise the existence of a common pattern in several groups of animals which are not otherwise very closely related (molluscs except cephalopods, nemerteans, platyhelminths, annelids); but overlying the basic similari- ties there are very many variations in detail, both in the early cleavages and in the types of larvae which are produced by early development. To keep within the limits of space available here, it will be necessary to attempt a ruthless simplification. The cleavage begins by two more or less vertical divisions, cutting the egg into four blastomeres, which are typically somewhat twisted in relation to each other. These four are conventionally known as A, B, C, and D. hi the next few cleavages, each of these cells remains as a fairly large macromere, and gives off a succession of smaller micromeres, the first group of which are known as la, lb, ic, id, the second group as 2a, 2b, 2c, 2d and so on. The cleavage spindles by which these divisions occur do not lie either vertically or horizontally, but at some angle between; and if the spindles are tilted to the left of the vertical in the formation of the first group, they will be tilted to the right for the next group. There is in fact an alternation from one tilt to the other. While the macromeres are giving off new rings of micromeres in this way, the already formed micromeres continue to divide in the normal way, again with their spindles tilted like those of the macromeres (Fig. 4.2, p. 61). This regular pattern usually continues for four division-cycles. The sub- sequent fate of each of these cells has been followed in detail, and we know exactly what organs each will form in the later embryo. There is no need to go into great detail about this here. Roughly, the macromeres form endoderm, and most of the micromeres ectoderm; but there are two spec- ial micromeres which produce the mesoderm, and which we shall fmd play a peculiarly important role in the mechanics of development. These are the cells id and 4J, both of which, as their designation indicates, are ultimately derived from the D macromere. Events after the initial cleavage vary a great deal in different groups. In many species a larva is formed; and this is usually some variety of the 'trochophore' type (Fig. 6.1). In the development of this, the ectodermal micromeres grow down over the macromeres, which bend inwards to form 94 SPIRALLY CLEAVING EGGS 95 a pocket-shaped primitive gut, v^hich gradually elongates until the whole embryo assumes a shape like an old-fashioned peg top. There is usually an apical tuft of cilia, and a strong ciliated band (the prototroch) more or less at the equator. Meanwhile the 4^ mesoderm cell has been covered over by ectoderm, lying in the corner at the boundary between ectoderm and endoderm. It divides into two, and each of these daughter-cells gives off a series of endomesoderm cells. In molluscs, these again become scattered, but in annelids they remain joined together as two long bands. Ectoderm Ciliated "Prolotroc. Blastopore Mesoderm stem-cells Mesoderm Figure 6.1 Gastrulation and formation of the trochophore larva in spirally cleaving eggs. A shows a section through a late cleavage stage, the micromeres lying directly on top of the larger macromeres, the slight space between them corresponding to the cavity of the blastula. Some cells from 2d and 4 J (shaded) have sunk beneath the surface. B, the micromeres spread over the macromeres, which are being drawn up into the embryo, forming the beginning of the primitive gut. C is an early trochophore ; note the two large cells, derived from 4^, each of which is budding off a row of meso- derm cells which extends round the blastopore between the ectoderm and endoderm. D, the trochophore is beginning to elongate, and the mesoderm bands are swung into a vertical position (this is typical of certain worm embryos, such as those o( Polychaetcs). A mouth has appeared, either as a new opening where the primitive gut has broken through the ectoderm, or in some forms by the blastopore becoming constricted into two. 96 PRINCIPLES OF EMBRYOLOGY These at first extend round the gut which is being pushed in fi:om the blastopore, but as the whole embryo elongates, the mesoderm bands come to lie more or less vertically. Finally a mouth is formed, either by the tip end of the gut breaking through the ectoderm to the exterior, or in some forms by a constriction which divides the blastopore into two. A trochophore larva of this kind is obviously only a minor variation from the general structure of a gastrula, and the whole egg takes part in its formation. Figure 6.2 Direct development (without larval stage) in a spirally cleaving egg (the freshwater oligochaete Ttibifex). The macromeres have cleaved to give a compact mass of largish endoderm cells {En), while most of the micromeres have formed small ectoderm cells lying on the other side of the egg [Ek). The cell 2d has, however, produced two groups of 'stem-cells', each com- prising one neuroblast and three secondary myoblasts ; these bud off a row of neural cells and three rows of ectodermal muscle cells. Below each of these columns of four are mesoderm-stcm cells derived from 4V/J<^ 'W^-^^mf r''i'\ ^W"^^"^ Figure 8.13 Regulative development in Drosophila. On left, a vestigial fly in which one mesothoracic bud has failed to evert and the other produced considerably more than half a thorax. (From Waddington 1953.) On right, conversion of eyes into palps following x-raying of the late larva. (From Waddington many of the imaginal bud cells are killed, and those that remain may pro- duce duphcated organs, or even something quite foreign to their normal fate (e.g. eyes in place of antennae or vice versa). Various authors then found that when the imaginal buds of the larva are cut into fragments which are allowed to develop in isolation, their behaviour is not strictly mosaic. Hadom and Gloor (1946) showed that the female genital bud be- haved like a series of overlapping fields (Fig. 8.14), and Hadom, Bertani and Gallera (1946) found that in the genital bud of the male considerable reorganisation and regulation of these fields is possible. Vogt (1946^) ob- tained very similar results with the eye-antennal bud. The same author (1946^) studied the development of eye-antennal buds of flies homozygous 142 PRINCIPLES OF EMBRYOLOGY for the gene aristopedia. This causes part of the antennal bud to develop into a leg instead of an antenna; and it was found that the amount of the bud which is diverted into this abnormal channel of differentiation can be altered by the temperature to which it is subjected during the third larval instar. One must conclude that the determination of the imaginal char- acter is still very labile until at least the time of pupation. Shatoury (1955) A ^ Figure 8.14 The female genital bud of Drosophila: a shows the genital organs of the adult; Ov, oviduct, at the top the two branches which continue to the ovaries have been cut through; R.S. receptaculum seminalis; Spt., sperma- theca; Ut., uterus; Vg., vaginal plate; yl.P., anal plates. The larval bud, lying across the intestine, is shown in b, and its position in the larva in c. The results of transplantations of fragments of the bud are summarised in d, which shows the overlapping fields from which the various organs are formed (shading corresponding to those of Figure a). (After Hadorn and Gloor 1946.) argues, on the basis of aberrant types of development found in certain mutant stocks, that the essential features of the imaginal buds are deter- mined by influences from the mesoderm which migrates into the buds during the third instar. Thus the determination of the buds which occurs in the embryo can only be of a preliminary and tentative kind. Even during the period of pupation, some degree of regulation is possible to the various imaginal buds. Waddington (1953) and Pantelouris and Waddington (1955) found that if one of the mesothoracic buds is THE INSECTS 143 removed, or if it fails to evert, the bud on the other side may regulate so as to form more than the half-thorax v^hich is its normal fate. Non-mosaic behaviour of a rather different kind is also exhibited by the gonads and genital ducts during the pupal period. Dobzhansky (193 1) first pointed out that if the testes of Drosopliila simiilans, which are nor- mally spiral in shape, fail for some reason to make contact v^ith the ducts which develop from the genital disc, then the spiralisation does not occur, and the testes remain ovoid. Stern (1941) studied the matter in detail, and showed clearly that the ducts induce in the testes the asymmetric growth which leads to the assumption of a spiral shape. In some species of Droso- pliila, the testes normally grow more or less equally in all directions, and thus remain ovoid ; and Stern found that if the larval testis of a species which should develop a spiral gonad becomes attached to the genital duct of a non-spiralising form, then it also fails to become spiral. This is one of the rather few cases in which a species difference is brought about by a difference in an inductive action rather than by being dependent on the nature of the competence of the reacting material. But, as Stem points out, we are dealing here with the transmission of an asymmetric growth stimulus and not with the evocation of histological type of tissue (Fig. 8.15). Figure 8.15 Interaction of gonads and genital ducts in Drosophila melanogaster pupae : a shows the outline of the testis at the time it becomes attached to the male duct; h, the normal coiled form it assumes; c, the uncoiled form resulting from failure of attachment. (From Stern 194 1.) Figure d shows the adult female genital tract in a fly from which one larval ovary was removed; the oviduct (arrow) to which no ovary becomes attached fails to elongate. (From Pantelouris 1955.) 144 PRINCIPLES OF EMBRYOLOGY The reaction between the testis and the ducts is not all in one direction, since pigmented cells migrate out from the testis sheath on to the duct, whose colouration is thus dependent on the kind of testis with which they come in contact (Stem and Hadorn 1939). A more drastic effect of the gonad on the associated duct occurs in females, where Pantelouris (1955) has shown that the lateral branch of the oviduct does not elongate unless it makes contact with the ovary. The ovary itself seems to be re- latively independent in its growth, and can, though rather rarely, attain its normal size even when not attached to any genital duct; this may occur even in a male host. SUGGESTED READING Most of the original literature on experiments on embryos is in German; Seidel 1936, 1952a recommended; Haget 1953 is a fine original paper (in French); Wigglesworth 1947 summarises some of the Hterature; see Poulson 1945 for chromosomal control. For pupal stages, Hadorn 1948^, Waddington 1942(1. For pupation hormones, Williams 1951, Wigglesworth 1954, Seidel 1952^. CHAPTER DC THE VERTEBRATES: THE AMPHIBIA AND BIRDS THE GROUP of vertebrates contains a variety of types which are sufficiently diverse to exhibit most of the important principles of embryology, but are not so bewilderingly various as to obscure the fundamental plan of which they are all modifications. They are therefore pecidiarly suited for comparative study. Moreover, the rather large size of many vertebrate eggs has made them favourite objects for experimental analysis, and our understanding of the epigenetics of tlie group is at least as great as that of any of the invertebrate phyla. This is particularly true of the Amphibia, and only slightly less so of the birds. These two groups have for long been classical teaching material, since frogs' and chickens' eggs are some of the easiest to obtain for students' use. The discussion of vertebrate development in this book will be largely based on these same two ob- jects, although as an amphibian type, the newt's egg will be referred to perhaps more often than that of the frog, since, although they are basically similar in the characters of interest in an elementary account, the former shows these features in a somewhat clearer way; moreover, for technical reasons connected with ease of manipulation, it has proved more favour- able than the frog egg for experimentation. It is only after the early development of these two types has been described and discussed that we shall turn to consider, more shortly, the embryology of the other vertebrate phyla. Detailed descriptions of the development of Amphibia are to be found in many general embryological textbooks, particularly Dalcq and Gerard (1935)- For the chick there are several special monographs. Patten (1950) and Huettner (1949) are good descriptive texts, the figures in the latter being particularly clear; Hamilton's (1952) revision of Lillie is the most complete descriptive monograph, but tends to neglect non-American work; Waddington (1952^) deals mainly with the early stages of develop- ment, and with experimental studies. Details of most microsurgical tech- niques are given by Hamburger (1942) and Rugh (1948), but neither of them deals with organiser grafts in birds. I. From the unfertilised egg to the formation of the blastula [a) The Amphibia The fully grown frog's or newt's egg is a fairly large spherical cell, some 2 or 3 mm. in diameter. It is usually seen after being extruded into the 145 146 PRINCIPLES OF EMBRYOLOGY water; normally the eggs are fertilised either as they leave the female's body (in frogs) or by sperm which the female has taken up into her cloaca (in newts). The egg within the ovary and oviduct is surrounded by a viscid layer of jelly, which swells on contact with water into a thick protective covering; in newts each egg is enclosed within a separate capsule, but in frogs a whole clutch of eggs coheres into a single mass within which the individual eggs are scattered. Amphibian eggs contain moderate quantities of yolk; much more than non-yolky types such as echinoderms or ascidians, much less than the extremely yolky birds' eggs. The yolk is present in the form of small platelets or ovoid granules, and, although scattered throughout the whole egg, is more concentrated at the vegetative end where the granules are also larger in size. Near the opposite, animal, pole is a large germinal vesicle fdled with clear sap. The heavy load of yolk granules makes it rather difficult to distinguish different regions of cytoplasm, such as those so well shovel in the ascidians, and it is only recently that the earliest phases of development are becoming clear (Reviews: Pasteels 195 1 ; Dalcq 1950&). There is, for instance, always a peripheral zone or cortex which contains little yolk; the animal half of it usually carries a considerable number of pigment granules, which make up a dark animal cap which is very clear in the frog; the outermost layer of all is a relatively impervious, very extensible membrane, the 'coat' (Holtfreter 1943a). hi the interior of the egg, it is usually possible after the germinal vesicle has broken down to distinguish a clearer animal plasm, in which the yolk granules are smaller and more scattered; this appearance may be due to the admixture of the nuclear sap (Fig. 9.1). In some forms (the frog and axolotl) there is also a darker 'marginal plasm' which lies in a circle fairly close under the surface j ust above the equator, and in the frog there is another, central, region with small yolk platelets (Pasteels 1951). The pattern of cytoplasmic regions in the unfertihsed egg is thus radially symmetrical and shows no obvious sign of the future dorso-ventral plane of symmetry. An indication of this plane does, however, appear fairly soon after fertilisation in many amphibian eggs. The first apparent result of fertilisation is a slight lifting-off of the inner or vitelline membrane from the egg, which thus becomes free to revolve within its jelly capsule and lie in its natural position with the heavy yolk-laden vegetative pole downwards; the swing round into this position takes only a few minutes. Soon afterwards, in the frog and some other species, a 'grey crescent' appears on one side, lying between the dark animal hemisphere and the pale yolky vegetative end. This is destined to play a fundamental part in later development. Its fate cannot be followed without special methods. THE vertebrates: the amphibia and birds 147 Z.VEG. Figure 9.1 Ooplasmic regions of the unfertilised egg oiXenopns. The main regions are': the animal region [Z.AN.); the marginal zone {Z.M.); the central region (AT.C.) and the vegetative region (Z.Veg.). The upper drawing shows the location of these in a transverse section, while the lower drawings illustrate the nature of the cytoplasms. (From Pasteels 195 1.) 148 PRINCIPLES OF EMBRYOLOGY since as the egg cleaves up into many cells, the even colour of the animal pole becomes broken up by the cell boundaries so that it merges into the paler tint of the crescent, v^hich ceases to be recognisable. But we can apply the technique of vital stained marks (p. 158). A small spot of colour placed in the centre of the grey crescent is found eventually to lie in the dorsal midline of the animal, and, in fact, somewhere in its notochord. The grey crescent therefore marks the dorsal side, and, in its position, corresponds to the grey chorda-neural crescent which appears, at a somewhat later stage, in the ascidian egg. The appearance of the grey crescent and the marking of the dorsal side is the first great step in embryonic development in the Amphibia. Natur- ally it is important to know its causal antecedents and its causal conse- quences; what brings it about, and what effect does it have? There is general agreement about the latter. After the grey crescent has appeared, every developmental performance of the egg is related to it. If, for in- stance, the egg is cut in half (which can be done even before the first cleavage by putting the egg into a loop of fme hair which is slowly pulled tight (Fig. 9.2), then only those halves which contain some grey crescent material will develop any of the main embryonic tissues, such as neural system, notochord, somites, kidney, etc.; ventral portions of the egg which have no crescent material, usually form only skin and disorgan- ised mesoderm and endoderm not recognisable as any definite tissue, though Dollander (1950) has recently shov^oi that in some cases a certain degree of regulation occurs and the ventral parts also produce a httle neural tissue and axial mesoderm. The grey crescent, in fact, is the pre- cursor of the 'organisation centre' of the gastrula, which, as we shall see, (p. 175) is the agent which causes the formation of the rest of the embryo. The antecedents of the grey crescent are less well understood (Ancel and Vintemberger 1948, Pasteels 195 1). Its position is certainly not completely fixed before fertilisation, since it is possible by suitable treatment to make it appear in any desired meridian of the unfertilised egg. If, for instance, an egg is fertilised with sperm brought on a needle to a given place on the surface, the grey crescent usually appears at or near the diametrically opposite side. Similarly, if the newly fertilised egg is held for some time so that one side of the yolky hemisphere is much higher than the other, the grey crescent usually appears on the higher side. The experiments of the last paragraph demonstrate that the grey cres- cent is not fixed before fertihsation, or indeed much before it actually appears. But the same experiments also suggest that there is a pre-disposi- tion of a certain plane to become the plane of symmetry. The experiments of tilting the egg, or of fertilising it in a definite place, do not always THE vertebrates: the amphibia and birds 149 determine the position of the crescent ; the cases in which they fail are those in which they do not overcome the existing predisposition. Again, in normal development there seems to be a tendency for the sperm to enter the egg in a predetermined plane, so that its influence on the position of the crescent usually reinforces a prior condition. Finally, when newly c Figure 9.2 A newt's egg, in its jelly capsule, is constricted into a dumb-bell shape soon after fertilisation. A, cleavage occurs first in the portion containing the nucleus; B, after some time a nucleus passes through the stalk, after which cleavage begins in the other (left) portion also ; C, both parts may give rise to a complete embryo. (From Schleip 1929, after Spemann.) fertilised eggs are constricted into two halves with a hair loop, we find that certain halves, although they contain the nucleus, develop into featureless lumps exactly similar to those, derived from a later stage,]which contain no grey crescent material; and this indicates that, even immedi- ately after fertiHsation, the crescent material is to some extent localised so that egg fragments may contain none, or too little, of it. 150 PKINCIPLES OF EMBRYOLOGY In recent times, Pasteels is the author who has studied in most detail the relation between the grey crescent and the various plasmatic regions of the egg (see liis review, 195 1). The internal contents of an amphibian egg are fairly fluid and if the egg is turned upside down and held with the vegetative pole uppermost, the heavy, yolky material from that end streams down and comes to lie against the cortex near the animal pole region. The grey crescent is not distinct enough in appearance always to be recognisable with certainty in such eggs, but as we have seen, in later development it gives rise to the blastopore and that structure, at least, is unmistakable. It is found that blastopores always appear at the edge of regions in which masses of yolky cytoplasm are in contact with the cortex (Fig. 9.4). At these blastopore regions invagination takes place and an embryo will eventually develop. Its cephalo-caudal polarity is determined by the gradient in yolk content, the future head end always originally lying nearest to the most concentrated mass of yolky cytoplasm. It is clear from these experiments that the position of the grey crescent is determined by the mutual relations of the yolky cytoplasm and the cortex. In eggs which have been held upside down and in which drastic altera- tions of the internal structure have occurred, it is difficult to be certain of the nature of the interaction which takes place between the yolky cytoplasm and the cortex. In the normal egg Ancel and Vintemberger have shown that the formation of the grey crescent involves a movement of the cortical layer of that region towards the animal pole (Fig, 9.3B). This seems to carry up with it some of the underlying yolky cytoplasm which thus becomes thoroughly mingled with the marginal cytoplasm with its larger content of basophylic granules and mitochondria. It seems probable that it is this mingling of yolky and marginal cytoplasm which is the essential feature of the grey crescent. If shortly after fertilisation and before the appearance of tlie normal grey crescent an egg is tilted slightly so that the animal-vegetative axis makes an angle with the vertical, the yolky cytoplasm slides down into the lowest position and in doing so leaves behind it a sub-cortical layer which has much the same appearance as that of a normal grey crescent : and as we have seen it eventually devel- ops into a blastopore. This experiment was one of the earliest to be carried out in amphibian experimental embryology. It was originally performed by Born in die 'eighties of the last century. It has recently been studied in detail by Pasteels (195 1). Pasteels also shows what happens when the experiment is carried out slightly later, after the appearance of the grey crescent. In eggs rotated at this time the blastopore will, again, eventually appear somewhere at the edge of the main mass of yolky cytoplasm, and THE vertebrates: the amphibia and birds 151 presumably it was preceded by the formation of something corresponding to a grey crescent, although it is not always possible to recognise this clearly. The position of the blastopore and of the putative grey crescent an. Figure 9.3 A. The effects of partial rotation of the uncleaved axolotl egg round an axis perpendicular to the dorso- ventral plane. The small arrow points to the grey crescent and the dots indicate the heavy vegetative ooplasm. Note that the blastopore always appears at the margin of this, in whatever position is nearest to the original grey crescent. (From Pasteels 195 1.) B. The movement of marks on the cortex towards the animal pole (P-a.) during the formation of the grey crescent. (From Pasteels 1951, after Ancel and Vintenberger 1948.) is now, however, not always determined by the direction in which the yolk slid down to the bottom of the egg. It is influenced rather by the position of the original grey crescent which had formed before the egg 152 PRINCIPLES OF EMBRYOLOGY was tilted. The blastopore appears at that edge of the yolky mass which is nearest to this position (Fig. 9-3^). Pasteels (see also Dalcq 1950/;) concludes that the properties of the grey crescent and thus eventually of the blastopore are brought about by the interaction of two factors. The first is a gradient in cytoplasmic constitu- RAN. P.VEG. RAN. RAN. Figure 9.4 The relation between the main yolk-mass (dotted) and the anterior-poster- ior axis of embryos developed following rotation of the frog's egg. The arrows point to the cephalic region of the mesoderm ; a is the normal situa- tion; b, c and d show inverted eggs in which there is little, considerable or complete redistribution of the yolk to lower pole. Note that the cephalic region of the mesoderm (i.e. the blastopore) always forms near the yolk- mass. (From Pasteels 1951.) tion which normally runs from the animal pole (rich in cytoplasm and cytoplasmic granules, and poor in yolk) to the vegetative pole (large yolk platelets and little cytoplasm). This gradient determines the cephalo- caudal polarity of the embryo which will develop. It interacts with a cortical field which has a point of highest activity in the future grey crescent region and falls off from that in all directions (Fig. 2.7, p. 42). It has been necessary to dwell at some length on these early events since THE vertebrates: the amphibia and birds 153 the determination of the plane of bilateral symmetry is, in many ways, of considerably greater importance than anything which happens in the much more striking phenomena of cleavage. Amphibian eggs, having a moderate charge of yolk, undergo cleavages which are definitely, but not exaggeratedly, unequal. The first cleavage plane is vertical, and usually, though not always, coincides with the plane of bilateral symmetry run- ning through the middle of the grey crescent — a coincidence brought about by the fact that the sperm has an influence, again considerable but not quite always effective, on the plane of the first cleavage, just as it has on the plane of the grey crescent. The second cleavage is also vertical, and perpendicular to the first. In many species, the first two dorsal cells are smaller than the two ventral ones, which is an indication that the cleavages are based on a bilateral-symmetrical pattern, which seems to underlie all vertebrate cleavages, although it is often difficult to distinguish. The third cleavage is horizontal, and the furrow lies above the equator, so that the animal cells are smaller than the vegetative. This is the first indication of the effect of the yolk gradient, whose influence is predomin- ant throughout the remainder of the cleavages. These soon become irregu- lar, and proceed faster in the animal than the vegetative region, so that the difference in volume of the cells becomes progressively more marked. At an early stage — about the fourth or fifth cleavage — a space appears in the centre of the mass of cells, the so-called cleavage cavity or blasto- coel. This, of course, lies above the equatorial plane, and, as the unequal cleavages proceed, it not only increases in size, but shifts further and fur- ther towards the animal pole. Cell division continues throughout the whole of embryonic develop- ment, but the 'period of cleavage' is considered to end when something else begins to happen. The first definite event which occurs to terminate it is the appearance of the blastopore and the beginning of gastrulation. By this time the egg, which at this stage is called the blastula, has become a hollow ball, with a thin roof of animal cells covering a large cleavage cavity or blastocoel, beneath which lies a floor of large yolky blastomeres. The most important processes which have been going on under cover of the cleavages have been two. Firstly, the divisions have cut up the egg into cells of a size more attuned to that of the nuclei; and there has been a considerable increase in the total amount of nuclear material, and perhaps a synthesis of DNA, to assist in. bringing nucleus and cytoplasm back to their normal relations (but see p. 58). Secondly, there has been a considerable movement of material ; if vitally stained marks are made on the vegetative pole of the egg, the dye is gradually carried right into the body of the egg, and eventually reaches the floor of the blastocoel. 154 PRINCIPLES OF EMBRYOLOGY The significance of this movement is not yet fully understood (Nicholas 1945). It does not, however, affect the very thin coat which forms the actual surface of the egg. This material, which has special properties of elasticity and toughness, remains on the exterior surface forming the boundary between the egg and the external medium, and seems to play an important part in the biophysics of the morphological changes which lead to the formation of the embryo (p. 439). The first sign of gastrulation is the appearance of a shallow groove, the blastopore. This lies somewhat below the equator and within the area of light-coloured yolky cells. As was said above, vital staining shows that it appears in the region of the egg derived from the grey crescent, which by this time is no longer recognisable. Before describing the later events, we shall foUow the development of the bird embryo up to the corresponding stage, so as to be in a position to compare the gastrulation of the two forms. [b) The birds A discussion of the development of the bird's egg up to the blastula stage will take much less space than was required for the amphibian. This is not because the events are less complex, but because our knowledge of them is less complete. The early stages of avian development remain difficult to explore, partly because the enormous stores of yolk obscure any cytoplasmic differentiation there may be, and partly because the egg at this stage is out of easy reach within the body of its mother, who does not lay it until the cleavage period is finished and the gastrulation begun. The true ovum of a bird such as the chick does not make up the whole of what we usually call the egg, but only the so-called yolk. This is covered with a well-defined and tough membrane — the vitelline mem- brane— outside which lies the albumen or 'white' which is again enclosed in a membrane, the whole being finally covered by the shell. All these parts, from the vitelhne membrane outwards, are non-living additions to the egg-cell, serving as sources of nourishment or means of protection ; they are laid down around the ovum after it has left the ovary, been fertil- ised, and is on its way down the oviduct. The 'yolk' or true egg-cell is not adequately described by its popular name, since although it obviously contains a very large quantity of yolk, this is by no means the whole or the most important part of it. At one point (which lies in the plane of the smallest section of the ovoid egg) there is a small area of clear cytoplasm containing the nucleus. It is from this that the whole embryo is derived. The great stores of yolk affect development from the very beginning. Whereas in most eggs, the penetration of one sperm suffices to prevent THE vertebrates: the amphibia and birds 155 the entry of a second, in highly yolky eggs such as birds' this mechanism breaks down, and a number of sperm penetrate. Only one of these com- pletes fertilisation by fusing with the egg nucleus; the remainder form subsidiary nuclei which probably play a part in digesting the yolk in the very early stages of development. In the next events, those of cleavage, the influence of the yolk is even greater. The cleavage furrows start in the clear cytoplasmic region, and never succeed in forcing their way down into the inert mass of yolk. The first two cleavage planes are vertical, but the third, while also starting as a vertical furrow in the flat layer of cyto- plasm, soon curves round so as to run horizontally parallel to the surface, and thus cuts underneath the cytoplasm and separates it from the yolk. The cleavage pattern, even in these very early stages, is irregular, and we have httle exact knowledge of how the cleavage planes are related to later development. The cleavages convert the cytoplasmic region into a small compact plate several cells thick. The cells contain fairly large quantities of yolk granules, and at the edges the plate merges into the uncleaved yolk through a zone of increasingly large and more yolky cells. Beneath the mass, however, the yolk begins to liquify, and in sections this region appears as an empty space, the 'subgerminal cavity'. There is considerable debate as to exactly what happens next; and on this turns the question of whether the subgerminal cavity is considered to be equivalent to the blastocoel cavity of the amphibian egg or as a mere local modification of the yolk. The thin plate of cells, lying on the massive yolk, is very easily shrunken and distorted by normal histological methods of fixation and presents great difficulties to the experimentalist; it will not be until ways are found of overcoming these that we shall reach a fully satisfactory interpretation of events. At present, one of the main views (held by Pasteels 1936-7, Peter 1938 and others) is that the subgerminal cavity is not equivalent to the blastocoel, but that the latter soon begins to appear in the form of irregular horizontal sphts within the mass of cells; these cavities gradually expand and run together until they form a thin space separating an upper from a lower layer. This space would then be the blastocoel, and the lower layer of cells would correspond with the large yolk-laden cells at the vegetative end of an amphibian blastula. In the bird, these authors hold, the lower layer merely stays where it is during gastrulation and forms the endoderm (Fig 4.1, p. 59). Others believe that the endoderm, instead of merely splitting off from the upper layer, is derived from it by a more active process which can be regarded as a modified invagination ; and they therefore consider the sub- germinal cavity, into which the endoderm is pushed, as a true blastocoel. 156 PRINCIPLES OF EMBRYOLOGY Different adherents of this view, however, have very different ideas as to the nature of the active process by which the endoderm originates. Perhaps the simplest of these is that of Patterson (1909), who supposed that the posterior edge of the cellular plate becomes folded under and grows forward below the remaining part. This has become one of the commonest accounts given in textbooks, perhaps because of its apparent simphcity; but, unfortunately, the evidence for it is negligible, and all authors who have examined the matter for the last forty years (except Lutz 1953, 1954) have denied it. Another, more plausible, view is that isolated cells are pushed out from the cellular plate and gradually build up a lower layer, Jacobson (1938) believes that endoderm formation begins in this way, but that the process goes on fastest in the posterior region (though not quite at the posterior margin) and eventually attains such an impetus there that the whole plate is bent down into a groove and migrates en masse into the endoderm. He describes the formation of a centre of invagination which would really merit the name of a blastopore and closely resembles the structures which, we shall see, are undoubtedly formed at a much later stage when the invagination of mesoderm occurs. Most later authors have been unable to confirm Jacobson's account in full. A final view (Hunt 1937) which must be mentioned is that, however the lower layer of cells is produced at this stage, it eventually does not form the endodermal organs of the embryo, such as the gut, but is at a later stage pushed out to the sides by cells which come out of the upper layer (from the primitive streak, which forms there, see later). Although it is not possible to decide fmally between these possibilities at the present time, the safest view is probably that the greater part of the endoderm is formed in the first way mentioned, by a splitting or delamina- tion of the original cell mass. On this interpretation the blastocoel is not represented by the subgerminal cavity, but by the cleft which separates the upper from the lower layer. During the period when the lower layer is forming, the mass of cells is also becoming thinner and spreading more widely over the surface of the yolk. From tliis time on it is usually knowoi as a blastoderm or blastodisc, and its upper and lower layers are frequently — and non-commitally, a useful point in the circumstances — referred to as the epiblast and hypoblast respectively. Moreover, a certain differentiation is appearing in plan, hi the more central part, the cells are beginning to have used up their content of yolk granules, so as to become more transparent, while all round the periphery they remain heavily charged and opaque. As the subgerminal cavity becomes more defmite below the central region, this differentiation into two concentric zones increases until there is a well- THE vertebrates: the amphibia and birds 157 marked central area pellucida surrounded by an area opaca. The embryo is formed entirely within the former. The opaque area is concerned mainly with digesting and liquefying the yolk, a process which is carried out chiefly by the underlying cells, which in this region do not separate cleanly from the upper layer, so that the whole zone remains a mass of rather spongy tissue not clearly divided into an epiblast and hypoblast till somewhat later. By the time the egg is laid, in most birds (such as the chick), the blasto- derm has already differentiated into a fairly well-defined area opaca and area pellucida, and, in the latter, the hypoblast and epiblast are well separated from one another, except perhaps in the anterior region, where the hypoblast may not yet have appeared. Very shortly after this, a thickening appears in the posterior region of the area pellucida. Tliis is the beginning of the primitive streak, and the first sign of gastrulation. 2. Gastrulation: presumptive maps At the beginning of gastrulation, the embryos of Amphibia and birds present completely different appearances. The former is a hollow sphere, with a thin roof and a thick floor surrounding a large blastocoel cavity; the centre of gastrulation is indicated by a blastopore, at this stage a small crescent-shaped groove lying within the area of pale yolk-laden cells of the blastocoel floor. The bird embryo is in the form of a blastoderm, a thin sheet of cells floating over a subgerminal cavity filled with liquified yolk; the blastocoel is represented only by a narrow cleft within the sheet, dividing it into an epiblast above and a hypoblast below; and the centre of gastrulation is indicated by the 'primitive streak', a short linear thicken- ing in the posterior region of the area pellucida. The end-products of gas- trulation in both forms are, however, similar in many essential respects. Both contain three layers of tissue, a mesoderm lying between an outer ectoderm and an inner endoderm. In both an embryonic axis is beginning to develop. An axial strand of ectoderm is thickening and folding up into a tube to become the rudiment of the central nervous system. Immediately beneath this, mesoderm is forming a notochof tht Jong narrow rod of tissue; while on either side of the notochord the ther of mesoderm is con- densing into a row of separate more or less cbf val blocks, the somites. And beneath the mesoderm again, the endoderdc is also foriTung an axial tube, the rudiment of the gut. The movemei^a-. and foldings by which these two dissimilar starting-points can be.orought to these two similar end-products must necessarily differ in Jiy ways; but there is one further point of resemblance which has not yet been pointed out, and which renders the two forms much more easily comparable and understandable. 158 PRINCIPLES OF EMBRYOLOGY This lies iii the maps of presumptive^ fate; if we take the centres of gastrulation as our points of reference, these have a similarity which the crude comparison of the blastula and the blastoderm does not exhibit. Amphibian eggs were the first to which the technique of vital marking was systematically applied to discover the map of presumptive area. The method consists in pressing against some region of the egg a small lump of agar impregnated with a dye which will colour the cells without doing them any great damage. The coloured patch can then be followed through Figure 9.5 The movement of vitally stained marks towards the blastopore, and thence into the mesoderm ; e shows the location of the material in a transverse sec- tion of the neural plate stage. (From Vogt 1929.) its later development, and its fate ascertained (Fig. 9.5). This was done by the German embryologist W. Vogt in 1925, and his results, summarised in 1929, have remained substantially unchanged ever since, although many later authors hc^^^ i^ vised them in detail (Fig. 9.6). Vogt showed that '^^7 whole lower part of the blastula becomes endoderm and forms tB^<^'ut and its annexes ; the whole upper hemisphere becomes ectoderm, diffe Vitiating into the nervous system and the epider- mis with its derivatives ^uch as the ears, nasal placodes, etc. In between these two lies a broad belt w^ -^h forms the mesoderm; on the dorsal side, ^m.. ^ The word used by the German authors who originally made such maps was *pra- sumptive', and many English authors use 'presumptive' for this: American writers, however, tend to use 'prospective*. THE vertebrates: the amphibia and birds 159 immediately above the blastopore (and thus in the location of the old grey crescent), this develops into notochord; below this region, and further to the side, lie two areas which become the two rows of meso- dermal somites, while the remainder of the belt forms the rest of the mesoderm (side-plate, tail-bud, etc.). Thus, if we look at the map from the blastopore outwards, we fmd first a zone of endoderm within which the young blastopore lies, then a ring of mesoderm, and fmally the ecto- derm. The elucidation of the presumptive map in the birds was by no means so simple technically as in Amphibia (summarised: Waddington 1952^). Figure 9.6 Map of the presumptive areas of a urodele gastrula. The blastopore Bl lies in an area of endoderm (in which the gill shts of the pharynx are indicated). Immediately anterior to it is a wide region of presumptive notochord (dotted), and on either side of that somitic mesoderm (lined), which passes off into ventral mesoderm (unshaded). The animal half of the egg is taken up by presumptive neural plate (dashed), and epidermis (unshaded). On the left, seen from the side, on the right from the dorsal surface. (From Pasteels 1940.) A beginning was made by Wetzel in 1929, who was able to stain small regions of the blastoderm in the opened egg; Graper, at about the same time, made speeded-up stereographic cinema films through a window in the shell, from which similar conclusions emerged; and in the next few years, Waddington was able to check many of their suggestions by ex- periments made on blastoderms removed from the shell and cultivated in tissue culture. Improvements of the methods of vital staining were made by Pasteels (1936-7); and Spratt (1946) has developed a technique of marking parts of the blastoderm with carbon particles, which allows of a more precise labelling, but can only be done on embryos cultivated in vitro. The results of studies by vital staining in ovo and by carbon marking in vitro have yielded very different maps for the early gastrula, i.e. for the i6o PRINCIPLES OF EMBRYOLOGY Stage at which the streak is just forming. These are shown in Fig, 9.7. The main difference arises from the fact that Pasteels, in his work with vital stains, fmds that during the growth of the streak a considerable movement takes place towards the anterior, while this was not apparent in Spratt's studies. Pasteels' student Malan (1953) has recently examined the matter again, and it has been rather convincingly shown that the absence of this movement in Spratt's material is due to the abnormal conditions of the in vitro culture, to which the early stages are particularly susceptible. Thus Figure 9.7 Maps of presumptive areas in the chick. On the left, just before the streak appears, according to Pasteels, above, and according to Spratt, below (taken from Malan 1953 and Hamilton 1952 respectively). On the right tw^o stages in the formation of the streak (from Waddington 1952^). Epidermis, white; neural tissue, vertical lines; notochord, dotted; axial mesoderm, close horizontal lines ; lateral mesoderm spaced horizontal lines (the very widely spaced lines in Hamilton's map are extra-embryonic mesoderm) ; already in- vaginated mesoderm, crosses. although Spratt's map has been accepted by most recent American authors (e.g. Hamilton 1952, Patten 1950, Rudnick 1948), Pasteels' earher one is probably nearer the truth. In considering this early stage, we are faced, however, with another uncertainty. What is the presumptive fate of the very early primitive streak? Vital staining has shown that in its later stages the streak consists of presumptive mesoderm. But there are great technical difficulties in making critical experiments of this kind on tlie early stages, and it remains perfectly possible that there is some presumptive endoderm still remaining in the streak when it first forms; this would certainly be so if Jacobson's THE vertebrates: the amphibia and birds i6i account of endoderm formation (see p. 156) were accepted. There is little doubt that in reptiles (p. 234) the endoderm comes from somewhere in this general region of the embryo, and if in birds it originates from a defmite part of the surface of the original cell-plate, this must be the place; but, of course, if it arises entirely by delamination from the lower part of mass of cells, there would be no definite location for it on a presumptive map of the surface. We are on firmer ground when we turn to the other tissues. Vital marks have clearly shown that most, if not all, the primitive streak and the area on each side of it becomes mesoderm, while the area further away takes part in the formation of the ectoderm ; the prospective neural ectoderm lies anterior to the mesoderm near the midline. Comparing the maps of Amphibia and birds, one sees that their general pattern would be similar if one might suppose that the amphibian gastrula has been opened at some point within the area of skin ectoderm, and this hole enlarged until the original map was flattened out to a circle. We should then have an area of endoderm immediately round the blastopore, surrounded by a ring of mesoderm fringing which is an outer ring of ectoderm, with the neural ectoderm concentrated at one end, the anterior. Tliis is exactly the picture we should find in the early primitive streak stage of birds if we suppose that any endoderm originates from the surface. A fuller discussion of the relations between the two groups is given on p. 243 . 3. The gastrulation movements (a) Amphibia We have now described the state of affairs at the outset of gastrulation and given a sketch of the condition at the end of it. The process of gastru- lation consists in the set of movements and foldings which convert the former into the latter. It is clear that, since the bird and amphibian gastru- lae differ so considerably, while the early embryos possess roughly similar organs, the two processes must take different courses. These must now be summarised. Gastrulation in the amphibian is the simpler of the two. It has been followed in great detail by the vital staining technique, but for our purposes it is only necessary to consider the general outlines of events. Before the advent of the staining method, the gastrulation process could only be inferred by comparison of a series of fixed and stained prepara- tions of successive stages. It was natural to try to build up a picture in terms of foldings, delaminations and localised growth; and a series of technical terms, such as 'epiboly', 'emboly', 'invagination', 'involution', etc. were employed in this connection. The newer methods showed that l62 PRINCIPLES OF EMBRYOLOGY the process is actually quite different from anything which had been en- visaged. The most fundamental type of gastrulation movement is a flow- ing or streaming one, in which a piece of tissue is carried bodily into a new situation. With the recognition of this fact, the older terms were seen to be not very appropriate, and they have largely disappeared from the literature. An exception may be made for the word 'invagination', which originally meant a massive infolding of a sheet of tissue (such as was described in the infolding of the endoderm in echinoderms (p. 82)), but which is now often used to cover any process by which an originally outer layer is moved into an interior position. The most obvious change on the surface of the egg itself during the process of gastrulation is the growth, rounding up and fmal closure of the blastopore. When it first appears, this is a small somewhat pigmented depression lying beneath the equator within the endoderm. Fairly soon it enlarges laterally to form a short groove. This continues to grow longer, and its two ends curve round to a crescent shape, which passes on to a horseshoe and then to a closed oval. Although the blastopore originally lay wholly within the whitish endoderm, by the stage at which it has closed up to an oval, the more pigmented tissue of the animal hemisphere is found to be lying at its edge, so that the outside of the egg is completely dark except for the light spot of yolky cells within the oval blastopore; this is known as the *yolk-plug' and is a very characteristic and easily recognisable feature. The history of the blastopore is, however, by no means complete; it gradually contracts in area, drawing together above the yolk-plug which is fmally covered over and hidden from sight. By the time gastrulation proper is ended, and the first signs of the embryonic axis are appearing, the blastopore has been reduced to a narrow slit which runs in the direction of the embryonic axis. By following through the history of vital marks, we can see that much more has been happening than the appearance of the blastopore would lead one to expect. Marks made anywhere within the ring of prospective mesoderm are seen to move down towards the edge of the blastopore, to flow over it, and, as can be shown by later dissection, to move away from it again underneath the surface. The lips of the blastopore are, in fact, not fixed positions occupied by a definite tissue, but are mere structural appearances showing where the flow of tissue along the surface turns downwards towards the interior of the egg. This explains how the lip, which was originally made up of the pale endoderm, later becomes lined with the darker material of the animal hemisphere; the pale material has already moved away inside, and the dark material has streamed down to replace it. THE vertebrates: the amphibia and birds 163 The streaming movement towards the blastopore begins when the blastopore is quite small; it always goes on fastest in the region in which the blastopore first appears, and the movement here involves a great stretching and elongation of tissue in the direction of the meridian joining the blastopore to the animal pole. This meridian will become the mid- dorsal line of the embryonic axis when this begins to form; and this part of the blastopore is, therefore, known as its dorsal lip ; the ventral lip is the last-formed portion which eventually appears on the diametrically opposed side of the yolk-plug. The material which flows in round the dorsal lip elongates considerably while doing so, and becomes narrower from side to side; this means that material invaginating further laterally Figure 9.8 The gastrulation movements in a urodele, seen from the side. has to move in towards the midline. The invagination streams, therefore, converge from the sides towards the middle, as shown in Fig. 9.8. More- over, as the ring of prospective mesoderm moves into the interior of the egg, its place at the surface has to be taken by the prospective ectoderm. We shall see later that the prospective neural ectoderm also elongates along the midhne, narrowing as it does so ; thus, the whole of the dorsal convergence has to be compensated for by a lateral expansion of the prospective skin. In its crudest outline, therefore, the gastrulation move- ments of the animal hemisphere of the egg can be summarised as, firstly, a great elongation and narrowing along the dorsal meridian (the elonga- tion being so much that the material flows round the blastopore and finishes up as a double layer), and, secondly, to compensate for this, a lateral expansion, in a plane at right angles to the dorsal one, of the pro- spective skin at the opposite side of the egg ; with, of course, one process l64 PRINCIPLES OF EMBRYOLOGY changing smoothly into the other as one goes from the dorsal midline towards the sides. The process we have described so far would give rise to only two layers, an outer which is the ectoderm and an inner which is the mesoderm. Within this again lies the endoderm, and we have as yet said little as to how it gets there. As a matter of fact, it is the endoderm which starts the whole movement, since the early blastopore lies wholely within it. At this stage, the invagination movement of the endoderm consists in the withdrawal inwards of the main bodies of some of the large endoderm cells, a process which can only be seen in sections (Fig. 20.13, P- 444). A little later the withdrawing endoderm is followed by the first mesoderm flowing round the dorsal lip. As this movement of the mesoderm spreads to more lateral regions, making the lateral lips of the blastopore, the edge of the mesoderm separates from the endoderm to form a free margin such as is pictured in Fig. 9- 10; only in the mid-dorsal line is the comiection per- manently retained at the anterior of the archenteron, and here it has never been easy to decide where to draw the boundary between meso- derm and endoderm. The separation only works round slowly to the ventral side, with the gradual spreading of the blastopore lips. Meanwhile, the region of endoderm which has become free of the mesoderm behaves as though it were sucked in to the interior, folding inwards and at the same time elongating along the dorsal meridian to keep pace with the mesoderm to which its anterior end is still attached. Along the dorsal surface of the endoderm a groove appears, at first shallow, but gradually becoming deeper. This is the primitive gut, or archenteron. As is clear from a section (Fig. 9.9) its walls and floor are made of endo- derm, but its roof is at first mesoderm — in fact, the mid-dorsal mesoderm which will later diflerentiate into the notochord. Gastrulation is often said to be completed by the time the blastopore is reduced to a small sht. Although this is not actually the case, as can be seen from Fig. 9.10 (see also p. 263), it will be as well to pause in our accoimt of it to notice some of the more obvious changes which begin to occur at this time. When the yolk-plug finally disappears from view, the egg is still spherical in shape, evenly coloured all over with the darker tint characteristic of the ectoderm, and diversified only by the blastopore sht, which is elongated in the meridian of the dorsal axis. Fairly soon after this, the first signs can be seen of the differentiation of the ectoderm into the neural system and the skin. The neural area lies immediately in front of the blastopore, and first appears (for instance in the newt, in which it is well exhibited at this stage) as a pear-shaped or dumbbell-shaped area of some- what darker colour. The edges of tliis area soon become elevated as ridges, THE vertebrates: the amphibia and birds 165 while between them the surface is somewhat flattened to make a wide shallow depression. The ridges wliich mark the boundary of the area are known as the neural folds, while the depressed area between them is the neural plate. Sections show that the neural plate is thicker than the remainder of the ectoderm (Fig. 20.21, p. 452). As time goes on this thickening increases, the plate simultaneously becoming narrower and the folds higher, until the whole neural area becomes more appropriately referred to as the neural Figure 9.9 Semi-diagrammatic drawings of newt gastrulae sectioned through the dorso- ventral plane. The ridge on the wall of the archenteron inc, d and e shows where the endoderm and mesoderm are separating from one another. (From Spemann 1938.) groove rather than the neural plate. Eventually the neural folds approach so closely that their upper margins touch and fuse with one another; neural material joins on to neural, and skin to skin, so that the neural groove be- comes converted into a tube lying beneath an unbroken covering of epi- dermis. Clearly the tendency of material in this region of the egg to converge towards the mid-dorsal line, which we noticed during the gastru- lation movements, has continued even after the stretching in length has become less marked. The same is true of the underlyuig mesoderm. The central strip which overlies the primitive gut and forms its roof condenses together into a single median strand, the notochord; while the tissue i66 PRINCIPLES OF EMBRYOLOGY which Ues shghtly more laterally also accumulates towards the midline, forming two strips of thickened mesoderm, which soon become seg- mented transversely, to form two rows of more or less cubical blocks, the somites. All these changes begin at or near the anterior end, and progress steadily posteriorly; though it should be noticed that the neural groove, being originally wider at its anterior end, does not succeed in closing over so early in its widest parts as it does somewhat further back. At the most posterior end of the embryonic axis, the remains of the blastopore persist Figure 9.10 Sagittal sections at three stages {a, b, c,) of gastrulation in a urodele, to show the expansion of the archenteron and the obliteration of the blastocoel ; the extent of the lateral mesoderm is indicated by shading. Below, J is a dorsal view on to the blastopore (presumptive notochord closely dotted, presump- tive neural system dashed); e is a yolk plug stage;/, the first appearance of the neural plate ;^, the neural fold stage. Note that in /considerable meso- derm, and even some notochord, is still on the surface, while even in^ there is a little mesoderm between the neural folds near the remnant of the blasto- pore. (From Pasteels 1940, after Vogt and Nakamura.) as a narrow slit; and here, as was hinted above, conditions are still much the same as they were in the more widely open blastopore of the yolk-plug stage, and gastrulation movements still proceed, although on a smaller scale. The notochord, at the dorsal apex of the blastopore, is the first to follow the endoderm completely below the surface, but the invagination of mesoderm round the lateral lips persists for some time longer. Vital staining demonstrates, in fact, that by the first appearance of the neural THE vertebrates: the amphibia and birds 167 plate only the first dozen or so somites have been invaginated, and the mesoderm of the greater part of the trunk, and the whole of the tail, is formed from the small area which still remains on the surface (p. 264). The formation of these organs involves a great increase in length, and the tendency to axial elongation in the midline takes on a new lease of life in this slit-blastopore region. The focus of this elongation is, according to Pasteels, not quite at the blastopore itself, but slightly anterior to it, at the most posterior limit of the invaginated notochord. {b) Birds Although the gastrulation movements in birds are more complex than in Amphibia, they are perhaps easier to visualise and to describe, since they occur not in a sphere, but in a flat circular disc. The main comphca- tion which is introduced is a double streaming movement along the mid- line, forwards at an early stage, and backwards later on. There is moreover another important difference from the Amphibia in connection with the type of growth which is proceeding. In the latter group the total mass of the egg is more or less constant during gastrulation; 'growth' consists in the divison of cells into smaller units, and the conversion into living sub- stance of yolk, already contained in the cells. In the birds, on the other hand, the greater part of the yolk lies outside the tissues, and during gastru- lation this is being digested and assimilated, so that growth involves an actual increase in the cellular mass. We have seen (p. 155) that at the beginning of gastrulation in the chick a lower layer of cells is already present, formed either by delamination or by migration from a posterior region of the blastoderm. This remains more or less in situ as the endoderm. It undergoes a general spreading out to cover the whole underside of the blastoderm, which may involve some posterior-to-anterior streaming, but if this occurs at all it is difficult to investigate by vital staining methods, and little is known about it. The upper layer of the blastoderm consists of the presumptive meso- derm and ectoderm. (For which reason it is sometimes referred to as the mesectoderm, but since this word has been used in other senses, it is better to call it the epiblast.) The formation of mesoderm begins at an early stage. The site of its formation is the primitive streak, the linear thickened area whose appearance in the posterior part of the blastoderm we have taken as the signal for the beginning of gastrulation. At the primitive streak there is no open channel leading from the outside to- wards the interior, such as one fmds at the amphibian blastopore. Never- theless, vital markings show that tissue which originally lay on the outer surface of the epiblast streams from both sides towards the streak, turns l68 PRINCIPLES OF EMBRYOLOGY downwards when it arrives there, and moves away again in a lower layer which lies beneath the surface but above the endoderm, and which is there- fore the mesoderm (Fig. 9.11). The first mesoderm to pass through the streak migrates, not only laterally, but also towards the anterior. It does not form part of the mesoderm of the embryonic axis, but finally lies well out to the side. This is in strong contrast to what happens in Amphibia, where the first invaginated mesoderm remains in the dorsal axis. But the difference is merely one of the place at which the invagination of meso- derm begins; in the Amphibia this is the dorsal lip, wliile in the birds the first part of the primitive streak is in a region corresponding more nearly to the ventral or lateral lips. — Epiblast . — Mesoderm — Endoderm Figure 9.11 Semi-diagrammatic section through the primitive streak of the chick, to show the invagination of mesoderm. Very soon after, or perhaps even simultaneously with, the beginning of this mesoderm-invagination, another movement affects the region of the primitive streak. This is a streaming forwards along the streak to- wards its anterior tip. As this movement gets under way, the streak length- ens, and pushes out across the area pellucida. Whereas when it first appears it occupies only a fifth, or less, of the diameter, it eventually comes to extend more than halfway across the still circular area. While the forward movement is still continuing in the anterior region, the posterior part of the streak starts to push out backwards. When this double movement is at its height, the streak is elongating so fast that it draws out the area pellu- cida from its original circular shape into an oval or pear-shaped form (Fig. 9.12). The movement bends the regions of the prospective map into long arcs lying on each side of the streak, and since the invagination is continuing all the time, by the close of the forward streaming nearly all the lateral mesoderm has disappeared in the anterior region, leaving the streak bordered by prospective somite material, with a small arc of pro- spective chorda at the most anterior end (Fig. 9.7, p. 160). The anterior end of the streak becomes somewhat more markedly thickened than the remain- THE vertebrates: the amphibia and birds 169 der, and a slight depression is visible in the centre of it. The structure has been given the name ofHensens node, and a rather exaggerated importance was attached to it in the earlier literature, since some authors held that it alone was the analogue of the amphibian blastopore; actually, we have seen that mesoderm is invaginated throughout the length of the whole streak. Hensen's node does, however, mark the site of a somewhat special type of invagination. In most of the streak during the phase of forward stream- ing, the direction of movement of the mesoderm has been in towards Figure 9.12 The rissue movements in the chick blastoderm : a, during the elongation of the streak; b, in the fully grown streak; c, during the regression of the streak. SoUd arrows show the movements on the upper surface, dotted ones those at a lower level (i.e. in the mesoderm). (From Pasteels 1940.) the streak from both sides, and out again in the lower layer towards the sides and somewhat forwards, as indicated in Fig. 9.12. The formation of Hensen's node seems to indicate the beginning of an invagination directed wholly along the midline; tissue, presumptive notochord to be exphcit, moves from slightly anterior to the node backwards into it and after sinking to the lower level, passes out again directly forwards, so that a tongue of notochord extends anteriorly to the tip of the streak. This is known as the head process. The formation of the head process brings to an end the forward move- ment along the embryonic axis, and from then onwards all the streaming movements are directed towards the posterior end. Although, as we saw, lyO PRINCIPLES OF EMBRYOLOGY the backward movement started at an earlier stage in the posterior region, it soon acquires relatively greater speed in the neighbourhood of the node, which therefore travels back down the streak, catching up, as it were, the regions posterior to it. The node marks the most anterior point where invagination is still proceeding, and as it moves backward along the streak, the area in front of it is occupied by neural ectoderm in the upper layer and already-invaginated notochord and somites in the lower. Since the node is moving faster than the more posterior parts, the total length of the streak is continually being reduced; but it is a long time before the node finally overtakes the posterior tip of the streak and thus obliterates it altogether. Before this happens the fundamental organs of the embryonic axis appear at the anterior end much as they did in the Amphibia. The plate of neural ectoderm rolls up into a groove and finally into a tube sunk below the epidermis; the underlying mesoderm separates itself into a median notochord flanked by thickened strips of somite material ; and these become transversely segmented to form the paired cubical blocks of the somites themselves. The remnant of the streak which persists in the posterior all this time, where invagination is still proceeding, may be compared with the slit-like blastopore which we saw remains active while the neural groove is forming in the Amphibia; but in the birds the structure is not only relatively larger than in the frog, but continues in being to a stage in which the anterior part of the embryonic axis is much further advanced. The formation of a definite gut from the endo- derm will be described later (p. 252). 4. General properties of gastrulation movements We have confined ourselves so far to a straightforward description of the movements which carry the regions of the early gastrula into their fmal positions. These are the fundamental events by which the future animal acquires its organic form, and the biophysics of the process will be discussed in some detail later (Chapter XX). There are, however, some general points which may be mentioned here. The forces producing gastrulation are not entirely functions of the egg as a whole, but are inherent in quite small parts of it. This emerges clearly from experiments in which parts of the gastrula are isolated. For instance, if in the stage with a fully formed primitive streak the chick blastoderm is cut transversely into two parts, the expected backward streaming along the axis takes place in both of them. This leads to the protrusion from the anterior part of a 'tail' containing the axial organs (neural tube, notochord and somites), while in the posterior half the medial material withdraws THE vertebrates: the amphibia and birds 171 posteriorly, leaving a gap (Fig. 9.13). The same phenomenon can be seen even in smaller fragments when these are grafted into abnormal situations. Grafts taken from the region of presumptive mesoderm in the newt and placed in some other part of the gastrula, usually succeed in moving below the surface into the mesodermal layer, often forming a small blastopore of their own to do so. Moreover, the direction in which this invagination occurs is more or less defmitely implicit in the fragment. Some of the most ■ , .fl!^:^^__ Q Figure 9.13 Tissue movements in parts of gastrulae: a shows two vegetative half- gastrulae of the newt placed together so that the blastopores point towards one another. The form which develops (b) is a'duplicitas cruciata'; the two streams of mesoderm from the two blastopores have met head-on, and been forced to spread out to each side, so that each head region is derived half from one egg and half from the other. (From Schleip, after Spemann.) Figure c shows the 'tail' developed from the anterior portion of a chick blastoderm transected just behind the node. (After Waddington 1932.) Striking examples of this have been described by Waddington (1941) in the anuran Discoglossus, in which the gastrulation movements are very rapid and perhaps for this reason seem to be rather definitely determined in the tissues ; if a small part of the dorsal mesoderm of this form is re- moved and grafted back, reversed in direction, it starts pushing out a tongue of tissue which moves in the opposite direction to that of the main stream of mesoderm which surrounds it. The inherent movement-tendencies of the parts of the gastrula are, however, at first by no means unalterable, as many experiments have 172 PRINCIPLES OF EMBRYOLOGY shown. For instance if two gastrulae are cut in half transversely and the two halves containing the blastopores combined, the two forward-moving streams of axial mesoderm meet one another before they have completed their elongation. As Spemann showed (cf. 1938) they then combine and spread out to the two sides, so that finally double embryonic axes are formed in a cross-shape (a so-called Duplicitas cruciata). Again, if a fragment is taken from a region where the movement is not very intense (e.g. the lateral or ventral mesoderm in the newt, or the more posterior parts of the primitive streak in the chick) and is grafted near a region of active movement (such as the dorsal lip of the blastopore or the anterior primitive streak), the inherent tendencies of the graft often appear to be swamped by the more powerful tendencies of its new surroundings. And this does not seem to be merely a question of the graft being physically swept along in the tissue streams of its new location, but the phenomena suggest that the graft is as it were infected with the characteristics of its neighbour- hood (Spemann and Geinitz 1927). This infection of a graft with the dynamic tendencies of its surroundings has an important bearing on the classical problem of 'the specificity of the germ-layers'. The older embryologists, relying entirely on descriptive methods of analysis, tended to reach the conclusion that ectoderm, meso- derm and endoderm were three fundamentally distinct types of tissue from a combination of which the embryo was built up. Soon after methods of experimental attack were discovered in Amphibia, however. Mangold (1925) showed that pieces of prospective ectoderm, if grafted into the mesoderm region in front of the blastopore, became invaginated with their surroundings, and thereafter behaved in every way as meso- derm, and also that ectoderm could be converted to endoderm in a similar way. In birds the demonstration is less complete, but Waddington and Taylor (1937) found that pieces of prospective ectoderm grafted into the primitive streak could become mesoderm, provided they became well enough assimilated to their surroundings for the coherent tissue to break down into single cells which migrate separately into the middle layer. These experiments show that there is no profound and permanent physiological difference between the three layers. SUGGESTED READING Vogt 1929 is a classical paper (in German, but the pictures should be studied). The early development of Amphibia is described in many texts (e.g. Nelsen 1953, Spemann 1928). Lehmann 1945, Fankhauser 1948, Dalcq igsob, Pasteels, 1951 add important information on early stages. For the chick, Waddington I952(j, Chapter 2, Hamilton 1953, Rudnick 1948. CHAPTER X THE EPIGENETICS OF THE EMBRYONIC AXIS I. Amphibia 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 import- ance 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 193 8, Dalcq 1941, Needham 1942, Lchmann 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 re- quired 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 early 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 unipoten- cies 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 teclini- cal flavour to conceal the fact that they suggest no explanation of any- thing. Moliere 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 ob- served in certain circumstances to become neural tube; and any possi- bility 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, cest 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 hap- pened 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 devel- oped 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 crucial region is in the dorsal vegetative quadrant v^hich 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. lo.i, 10.2). Grafts from the presumptive ectoderm, Figure io.i 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 195 1.) 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 con- clusively 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. ind.n.p. hnf 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 (/»-*Organl««r* Figure 10.12 The activation of the evocator. At the blastopore, something occurs which converts the inactive tissue into an active evocator (change of (£) 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 (£) 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 (e.g, by being grafted into the centre of presump- tive 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 Ipp Barth (1941) showed that the gastrula ectoderm of the axolotl (A. puncta- turn) developed sometimes into neural tissue when isolated in, as he thought, completely neutral salt solutions. This was a startling claim be- cause until that time it had always been held that ectoderm could only become neural if definitely induced to do so, and Earth'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 ecto- derm is particularly sensitive to abnormal external conditions and reacted by neuralisation to salt solutions wliich 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-defmed nervous organ. This organ always belongs to the anterior end of the brain (the forebrain or archencephalon). hi considerable con- trast 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 wliich 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 ecto- derm 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 (1940^^) that, if the competence of the gastrula ectoderm is set on so fme a hair-trigger that any of a number of stimuh 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 evoca- tors, and would have to confme our attention solely to what we can dis- cover as to the processes going on in a reacting ectoderm. As it is we can fmd important clues to further understanditig not only in the ectoderm 200 PRINCIPLES OF EMBRYOLOGY itself 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 un- doubtedly of extreme biological activity, should have a higher rate of respiratory metabohsm 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 metabohsm of the organisation centre at the time it exerts its main in- ductive 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 Linderstrom-Lang that this requirement was fully met. Using this instrument Needham and his co-workers (see Boell, Need- ham and others, 1939) found that in most series of experiments there was no appreciable difference between the rate of oxygen uptake by the blasto- pore 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 less- sensitive 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 acti- vity 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 BoeU are: Q'og (= ni^l, O2 per [xg nitrogen per hour) 49 for presumptive neural plate near animal pole, 21 for dorsal hp, 28 for posterior presumptive ectoderm, 1-3 for endoderm. There is, of course, more yolk in the endo- derm 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). BoeU 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 o Figure 10.13 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.) (1953^) also fmds 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. Fhckinger (1954) fmds that the rates of incorporation of radioactive CO2 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 CO2 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 COg output). They found that in the blastula roof the respiratory quotient is about 075. By the mid-gastrula, this has risen to i in the blastopore region, but is still only about 08 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 metabohsm is involving the breakdown of carbohydrate L- LIQUID! -' ^^, ,^ LIPOID --SOLID PERCENTAGE YOLK - -SOLUTION- SALTS, PROTEIN ETC. PRECIPITATED .- PROTEIN __ PIGMENT I HYALINE LAYER PIGMENT!! YOLK '02 80 60 40 20 0 1 7 1 .. , _. - / - / / - / / 1 20 40 60 80 'active' material Figure 10.14 On die left, a diagram of the stratification of gastrula tissues following high speed centrifiigation. 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. Woerdemaim (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 metaboHsm 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 breakdov^Ti of glyco- gen provides in the main the energy which must be utiHsed in the per- formance 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 (e.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 inliibits 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 tiU 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 (see Barth and Barth 195 1) is studying the mechanism by which the embryo utihses the energy derived from glycogen, and also that made available when the yolk is digested at a later stage. He finds that high- energy 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 metaboHsm of the egg, and again certain of them appear to go fastest in the blastospore region. A fact which emerges from cytological observa- tion 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 1952^). They occur more or less simultaneously throughout the animal cells, but more slowly in the endoderm, where the nuclei re- main large. Sirlin and Waddington (1954) found, in autoradiographs of 204 PRINCIPLES OF EMBRYOLOGY embryos treated with radioactive amino-acids, that these are incor- porated'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^imder way, there seems to be an increase in the cytoplasmic RNA. This can be demonstrated by measuring Figure 10.15 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 culti- vatedin a solution containing radioactive methionine. The amino-acidhas been incorporated into the proteins, and probably the nucleic acids, particularly in the nuclei of the blastopore region. (After Sirlin, I955-) 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 1952^). 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 invagina- tion 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 (1952^7, h) 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 in- corporation 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 metabohsm of this amino-acid seems to be of considerable importance, since administration of the unnatural analogue etliionine, which would be expected to interfere with the utiUsation of methionine, causes con- siderable inhibition of development. Herrman (1954) has described rather shghter 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. p 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 metabohc 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- bohc 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 hp, 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 metabohsm 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 deter- mination 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 unhydrohsed tissues, Clayton (1954) has found considerable differ- ences 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 show 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 incor- poration into the proteins of amino-acids labelled with radioactive isotopes. Eakin, Kutsky and Berg (195 1) have found that such amino- acids 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 Wad- dington (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 pro- THE 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 con- cerned with the development of species specificity; it is a well-knoMOi, 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. Neverthe- less 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 sub- stances as development proceeds. References to most of the recent literature will be found in Woerdeman (1953), Cooper (1950) and Clay- ton (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 Fhckinger and Nace (1952) prepared their anti-sera by injection of embryos, or parts of them. Clayton made anti-sera against (i) 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 in- stance, 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 wliich 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 develop- ment 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 (i) 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 (E^ 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 anti- gens also appear during neurulation, e.g. a fraction named Y (Fig. 10.16). Blastula Gastrula q Neurula Tail-bud Ectoderm Neural plate E'C part of 0 ? 1 CO EE' / Neurula Ectoderm EO part of C & £' ? y CEM \ Mesoderm \ CO MM' Unallocated X consists of C E and perhaps ot 'M' hers Y not further studied Z not further 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 synthes- ised during gastrulation, and it seems likely that immunological methods w^ill be a very pow^erful 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 m 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 epider- mis, 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 differentia- tion 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 sup- pressed the development of the neural tube. We have then clear evidence that soon after their determination, and during the early phases of differ- entiation, the different tissues produce different antigenicaUy-active chemical substances, presumably protein in nature. In later work, Ebert (1953^) used sera prepared against the protein (myosin) of adult chick hearts to test various regions of the early blasto- derm. 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 fuUy 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, hi the next stage, of the long primitive streak, Ebert tested not only whole blastoderms but parts of them, as showoi 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 Figure 10.17 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 1953^.) 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 211 entirely from the anterior region of the blastoderm. By the next stage (head-fold and early neural groove) it has disappeared also from the anter- ior 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 regional- isation proceeds within the region, disappear, or at least become un- detectable, 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 rudi- ment, 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 im- portance for our understanding of epigenetic processes. It seems quite compatible with the hypothesis (advanced on p. 407) that in develop- ment 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 A to 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 Gate 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 (1954^ and Clayton and Feldman (1955) have recently intro- duced 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 teclmiques on the problems of pro- tein synthesis in embryos, a considerable amount has been learnt about evocation by older methods. Brachet (1944, 1952^, 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 195 1); 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 begin- ning of gastrulation, particularly in the organiser region and in the invaginating mesoderm; a httle 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. 3 82). In both the heat- treated and the hybrid gastrulae the rate of oxygen consumption ceases to rise from the time at which development stops. [Barthand 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 wliich the induced ectoderm under- goes.] It is rather surprising to fmd that these changes can often be re- versed 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, 1948/)) showed that when gastrula ectoderm is submitted to abnormal media (producing so-called sub-lethal cytolosis) there is a massive appearance of microsome-Hke bodies in the cytoplasm. Waddington and Goodhart (1949) made much the same observation when studying the mode of action of the steroid-hke evocators. The location THE EPIGENETICS OF THE EMBRYONIC AXIS 2I3 of these substances iii 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 evo- cators the breakdown is accelerated. Finally, Pasteels (195 1, 1953) has noticed a similar appearance of basophilic cytoplasmic granules in gas- trula cells which have been centrifuged and thus caused to undergo spon- taneous 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 stimuh. They are probably in fact the site of the reactions h to cto d, etc. as postulated above (p. 197). This accords well with the evidence (pp. 90, loi) that centrifugable granules are important in determining the characters of the different regions in mosaic eggs, the gradients in echinoderms, etc. 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 symbol- ised as a in 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 Gothic 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 m 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 possi- bility is rendered improbable by the observation that if a porous mem- brane, 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, how- ever, as clear cut as one would desire to establish such an important con- clusion 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 estabhsh 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 sub- stances 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. Prehmin- ary 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 (1950I)) showed that, if yeast is labelled by being cultivated for some time in solutions containing phos- phorus-32, then dried and used as a graft in the gastrula, the radioactive material passes from the implant into the neurahsing 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 metabohsing tissues of substances released from the graft itself, which become available throughout the whole embryo. Again it is un- THE EPIGENETICS OF THE EMBRYONIC AXIS 2I5 clear whether these substances are in the form of individual aniino-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 v^hich 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 organ- specific 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 amino- acid, in which case it will yield httle information about the mechanisms of induction. Abercrombie and Causey (1950) have used radio-phosphorus to label regions of the cliick 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 shovwi 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 2l6 PRINCIPLES OF EMBRYOLOGY that evocation is dependent on specific rather than imspecific stimuh. 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 195 1), 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 1935^7, 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 hap- hazard. 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 deuter- encephalon). It is very thermolabile, being destroyed by heating to 90° C. and is insoluble in petrol ether. The adult tissues as used in im- THE EPIGENETICS OF THE EMBRYONIC AXIS 2I7 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 estabhshed by Toivonen's data. There are however several questions about them wliich 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 archencephahc evocator is a pure neural inductor while the spinal inductor is able to induce mesoderm as well as neural tissue, the anterior or posterior charac- ter 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, extremi- ties, 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 (193 4^) that the organiser material of the gastrula, when extracted with boiHng 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 sHght heat treatment. The abohtion of mesoderm-inducing capacity was confirmed and the evidence suggested that the neural- inducing capacity is not in any way regionally differentiated. Large masses of induced neural tissue sometimes form themselves into archencephahc 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 archencephahc evocator isolated by Toivonen from adult tissues. Lalher (1950) claims that the neural-inducing capacity of the gastrula organiser is abolished by treatment with formahn, but it is not clear whether this is true of Toivonen's archencephahc evocator. The mesoderm-inducing capacity of the hving 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 2l8 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 archencephahc 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 w^ith, substances which are active during gastrulation. Kuusi (195 1, 1953) has studied the evocatory pow^ers of various frac- tions (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 archencephahc (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. 7. Competence 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 1940(1, Holt- freter 195 1). 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 differentia- tion. Within each of these main categories 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 instabihty 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 1934^). 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 com- petence 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 pelhicida. 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 com- petence 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 equiva- lence of the treatments appHed 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 beheve that all competences throughout development can arise autonomously, without dependence on previous inductive processes. There w^ere 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 1940^). Another very interesting problem is that imphcit 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 varia- tions of this kind perhaps even tip the scales between the major alterna- tives 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 certamly 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 con- trolled 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 pheno- menon 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 he 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 character- istics of the species to which the competent tissue belongs (for discussion: THE EPIGENETICS OF THE EMBRYONIC AXIS 221 see Baltzer 1950, 1952a), 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 teeth (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 KJng (195 1) 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 evoca- tion has led us into the very heart of the complex metabolic hfe of the Mouth Sucker Figure 10.18 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 stimu- lating 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 ribo- nucleo-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 epigenetic 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. SUGGESTED READING The classical papers on organisers are Spemann 1918, Spemann and Mangold 1924. Spemann's owia 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 271- 89: Lehmaim 1945, pp. 203-350; Waddington 1952a, pp. 51-139. For biochemical aspects, Brachet 1944, Chapters 9 and 10, Boell 1948. Other valuable discussions, Brachet 1952a, b, Holtfretcr 19486, 195 1, Toivonen 1950. CHAPTER XI EMBRYO FORMATION IN OTHER GROUPS OF VERTEBRATES UNTIL fairly recently embryology has been in the main a comparative study. Its object has been conceived to be the derivation of a general scheme of development, of which the processes found in the different groups of animals could be regarded as modifications. In particular the diverse, but nevertheless obviously related, groups of vertebrates have provided fascinating material for study of the relations between different types of embryonic development. The wealth of available facts is indeed immense, and there are numerous textbooks devoted to it. Perhaps the best are Dalcq and Gerard's revision of Brachet's work and the recent book of Nelsen. Comparative embryology of what may be called the classical kind was historically a derivative of, and theoretically should include, comparative anatomy. The comparisons instituted were between particular stages of the embryos of the various groups, each stage being considered as a static anatomical form.The results of such study are of great interest in relation to the general processes of evolution, though we consider them nowadays rather as raising problems than contributing materially to their solution. From the point of view on embryology which underlies the writing of this book, this aspect of biology remains rather peripheral, and since an adequate treatment of it requires the citation of an enormous number of detailed facts, we shall have to omit it and leave it in the hands of the comparative anatomists. The comparative method is still, however, a valuable tool of analysis. It can be applied not only to anatomical data but also to information of the kind with which this book has been mainly concerned. Such applications have only recently been attempted, but there are two types of comparative embryology which one must expect to increase greatly in importance in the near future. One is the comparative study of dynamic anatomical processes. Among the vertebrates there is a body of material which most urgently calls for treatment in tliis way, namely the active processes of gastrulation in the various groups which have been revealed by the use of vital staining or marking. Secondly, we now know enough about the causal sequences of epigenetic processes in the early development of the various groups to begin to institute com- parisons between them, and to enquire what kind of alterations in these 223 224 PRINCIPLES OF EMBRYOLOGY causal mechanisms have been produced by the processes of evolution. The importance of the comparative method has been acknowledged in the main chapters dealing v^ith the vertebrates by treatmg iii immediate] uxta- position the two best-known types, the amphibian and bird. In this chap- ter the field of comparison will be widened to include the other vertebrate groups. These will not be treated so fully, since our purpose here is not so much to obtain a detailed and thorough knowledge of the epigenetic processes in each group but rather to provide a sketch of the salient fea- tures for purposes of comparison. Each group will first be discussed in turn, and in the last two sections of the chapter an attempt will be made to draw the parallels and point the differences between them. I. Cyclostomes and other primitive jish The eggs of a representative of the cyclostomes, the river lamprey [Lampetra or Petromyzon) have been studied in some detail in recent years. The egg is about 2 mm. in diameter. Its cleavage is total and very similar to that of the Amphibia. It gives rise to a blastula which is also very similar to the amphibian. At gastrulation a small knob begins to protrude in the spherical embryo and the blastopore appears just below it. The blastopore is small and remains so throughout the whole period of invagination and one sees nothing resembling the yolk plug of the frog. Weissenberg (1934, 1936) has studied the gastrulation by means of vital stained marks, and has derived a map of presumptive areas (Fig. ii.i). It shows a considerable resemblance to that of the Amphibia except that the axial mesoderm is, from the beginning, more concentrated towards the dorsal plane. According to Weissenberg, indeed, the presumptive Figure ii.i Map of presumptive areas in the young gastrula of the lamprey (cyclo- stomes). Close dots, chorda; spaced dots, mesoderm (somitic mesoderm with horizontal lines) ; close vertical lines, neural plate; spaced vertical lines, epidermis; w^hite endoderm. The position of the mesoderm on the ventral side is uncertain. (Modified from Weissenberg). EMBRYO FORMATION IN OTHER GROUPS OF VERTEBRATES 225 mesoderm does not extend right round the egg but on the ventral side the presumptive ectoderm comes into direct contact with the presumptive endoderm. This situation, which would constitute a considerable differ- ence from that of the Ampliibia, has been questioned by Pasteels (1940) who points out that in the early neural plate stage, when invagination is still proceeding around the blastopore, this structure is undoubtedly sur- rounded by a complete ring of mesoderm, some of which is being in- vaginated over the ventral lip at this time. Although Pasteels' material was not sufficient to enable liim to draw an alternative map of the presumptive areas in the early gastrula, he concludes that the mesoderm must in fact extend right round the egg between the ectoderm and endoderm. He suggests that the main difference between the gastrulation of the lamprey and the frog is that, in the former, the invagination of mesoderm over the lateral and ventral lips is delayed so that it does not begin until the main mass of endodermal material has already passed into the interior. The two processes in fact differ only in this comparatively minor matter of timing. Some attention has also been paid to the causal embryology or epi- genetics of these forms. It was shown as long ago as 1900 by Bataillon that if the first two blastomeres are separated each may give a complete embryo. The experiment has been repeated more recently (Montalenti and Maccagno 1935; Bytinski-Salz 1937^) and it has been shown that the situation is almost exactly the same as that revealed by constriction experi- ments in the Amphibia. Further, fragments from the dorsal lip of the blastopore can be grafted just as in nev^s (Bytinski-Salz 1937&; Yamada 193 8). When they come in contact with gastrula ectoderm they cause the induction of a secondary neural axis and, so far as experiment has gone, repeat in every way the behaviour we have seen in Urodele material. Unfortunately there has been little work by modern methods on other groups of primitive fish. Those types wliich have markedly telo- lecithal eggs and very unequal, but still total, cleavage would be particu- larly interesting as providing a transition to the higher forms, which are provided with very large masses of yolk and develop through the stage of a blastoderm. No vital stain experiments seem however to have been made on these transitional types. Ginsburg and Dettlaff (1944) bave shortly reported a small number of experiments on the embryos of the sturgeon Acipenser. This has an egg, about 3 mm. across, which is rather amphibian-like in the cleavage and early gastrulation stages. Grafts of the blastopore hp showed that it behaved as an organiser and could induce secondary neural folds in a host embryo. The donors from which the blastopore lip had been removed developed no nervous system or axial organs, and the authors suggest that the presumptive materials for these 226 PRINCIPLES OF EMBRYOLOGY are confined to a smaller area than in amphibian eggs, although presum- ably located in a similar position. The gastrulation process has been studied in some detail in the dog-fish, Scyllium canicula, a selachian and therefore usually considered more primi- tive than the sturgeons (Vanderbroek 1936). Its eggs, however, are more like those of teleosts (higher bony fish). They are provided with a large quantity of yolk, and the cleavage is partial, giving rise eventually to a blastoderm. This gradually spreads over the whole surface of the yolk, and as it does so invagination takes place along one sector of the circum- ference, after which the neural folds appear in the same position. The blastoderm is a fairly thick structure, and much of the endoderm lies from the beginning in the depths of it beneath the surface. This is particularly true of the extra-embryonic endoderm; some of the material which will form the endoderm of the embryo proper is at first on the surface, lining the position where the blastopore will appear. It is the first material to be invaginated. The disposition of the other presumptive areas is shown in Fig. 1 1.3. One point worthy of note is that the presumptive mesoderm does not extend right round the blastoderm on to the ventral side, but is confined to the dorsal region in the neighbourhood of the blastopore. 2. Teleosts The eggs of teleosts are rather large, and contain considerable quantities of yolk. Although there is extensive variation from species to species in the relative mass of yolk and of living cytoplasm, the cleavage is always par- tial and leads to the formation of a blastoderm. The structure of the egg at this time is not altogether simple. At the animal pole there is a relatively thick plate of well-defmed cells forming the blastoderm proper. Beneath this is a cavity, and underneath that again and extending for some distance around the blastoderm, the yolk is admixed with a fair quantity of cytoplasm and contains scattered nuclei; this syncytium is known as the periblast. Covering the whole surface of the egg is a thin cytoplasmic membrane which shows the properties of low permeability, high contract- ability, and lack of adhesiveness on its external face, which are also seen in the external membrane of the amphibian egg. It may be referred to as the 'coat', using the word employed by Holtfreter for the similar structure in Ampliibia. Where it lies over the yolk outside the limits of the blasto- derm, it is also knov^oi as the 'yolk gel membrane'. Its properties have been particularly studied by Devillers (1948) and Trinkaus (1949). The blastoderm expands in area and after a time the sub-germinal cavity below it becomes well marked (Fig. 11.2). The maximum depth of the cavity which is roofed by the thimiest region in the blastoderm, EMBRYO FORMATION IN OTHER GROUPS OF VERTEBRATES 227 is not at the centre but is eccentrically based. This provides the first marked sign of bilaterality in the developing embryo. Vakaet (1953) has, however, found that the oocytes of the fish Lebistes show a bilateral structure when they are growing in the ovary. It may well be, therefore, that the dorso- ventral plane in the blastoderm is foreshadowed at a much earher stage. Figure 11.2 A, B and C are lateral views showing three stages in the spreading of the blastoderm of the trout egg over the yolk. A', B', and C are views on to the blastoderm, which in the last two has been cut in order that it may be spread out flat. Bl, blastopore; e.a., embryonic area;^.r. germ ring. Gastrulation (Reviews: Pasteels 1940, Oppenheimer 1947) takes place by the in-rolling of the margins of the blastoderm. This occurs all round the circumference, but goes on most rapidly at one point, which becomes the site of embryo formation. The endoderm is the first tissue to be in- vaginated. Its presumptive area lies as a thin band in the margin of the blastoderm, much of it already below the surface (Fig. 11. 3). It is prob- ably concentrated mainly in the posterior region from which the embryo 228 PRINCIPLES OF EMBRYOLOGY will later arise, but its lateral and anterior extent is very inadequately known, and it may perhaps continue right round the whole margin of the blastoderm. Lying inside it, and the next material to be invaginated, is the mesoderm, which, according to Pasteels' work on the trout, cer- tainly continues round the whole margin. Inside this again, that is to say, towards the centre of the blastoderm, is the presumptive ectoderm. The shape of the presumptive neural plate described by Oppenheimer in Fundulus is quite different from that assigned to it by Pasteels in Salmo, but the overall disposition of the areas is much the same in the two forms and their differences are perhaps such as we might expect from two not very closely related species. Fundulu-s Figure 11.3 Trout Presumptive maps of the early gastrula (blastoderm) in the dogfish (Sela- chia), and Fundulus and the trout (teleosts). Endoderm, circles; cephaHc endoderm, small crosses; notochord, close dots; mesoderm, spaced dots (somites, horizontal lines); neural tissue, close vertical lines; epidermis, spaced vertical lines. (After data of Vandebroek, Oppenheimer, Luther, and Pasteels.) While the gastrulation is proceeding, the blastoderm is expanding rapidly, so as eventually to cover the whole yolk. Its margin is often tliickened, forming a structure known as the germ ring. After the expand- ing blastoderm has passed the equator of the egg, the germ ring contracts and acquires a superficial resemblance to a yolk plug before it covers the yolk mass completely. The extra-embryonic part of the blastoderm later becomes vascularised, and forms the yolk-sac by which the embryo absorbs its nutrients from the yolk. While the blastoderm is still expanding and gastrulation continuing at its margin, the embryo begins to appear (Fig. II .2). The connection between the embryo and the thickened germ- ring caused many of the earher embryologists to suppose that the two lateral halves of the embryo had originally been separated, one on each EMBRYO FORMATION IN OTHER GROUPS OF VERTEBRATES 229 side, as though the embryo had been spht from tail to head and the two halves pulled apart. These two half embryos were then supposed to come together and fuse in the midHne by a process to which the name concre- scence was given. It can, however, be seen from the map of presumptive areas that this is not the case. There is always complete continuity from one side to the other across the midline. The presumptive areas are wider from side to side than the defmitive organs will eventually be, but the movement from one situation to the other involves only a lateral contrac- tion and longitudinal stretching, and not a moving together and fusion of two originally separate rudiments (Fig. 11.4). Figure 11.4 Gastrulation movements in the trout : 1 is an early stage, showing the whole blastodisc; 2 and 3 are stages in the formation of the embryonic axis. Move- ments taking place in the surface layer shown in solid arrows, those occur- ring below the surface in dotted arrows. (From Pasteels 1940.) The mechanism of the gastrulation movements, and in particular of the spread of the blastoderm over the yolk, has recently been extensively studied by Devillers (1951^) and Trinkaus (1951). They both agree, in contradiction to certain earlier authors, that the spread of the blastoderm is not solely due to the contraction of the yolk gel layer. It seems probable that one of the essential factors is the activity of the periblast, which seems to have a spontaneous capacity to spread over the surface of the yolk. The blastoderm also has an autonomous tendency to expand, but it only does so over regions which the periblast has already covered and thus provided with a suitable substratum for it. 230 PRINCIPLES OF EMBRYOLOGY Quite a number of studies have been made on the epigenetic mechan- isms which bring about the development of the teleost (Review: Oppen- heimer 1947). They have dealt with several different species, none of which has as yet been fully investigated and the results do more to whet one's appetite than to provide a comprehensive account of the causal embryology of the whole group. The earliest stage at which operations have been made is immediately after fertilisation. Such work goes back to the experiments of Morgan in the 1890s. The results of the most recent workers on Fundulus suggest that any one of the first two, or of the first four, blastomeres can give rise to a complete embryo. For example, Nicholas and Oppenheimer (1942) ob- served embryo formation in sixty-five cases out of seventy-two in wliich they had eliminated one of the first two blastomeres chosen at random. There seems to be little sign at this stage of an organisation centre which is localised as is the amphibian grey crescent. The existence of such a centre is, however, claimed by Tung and Tung (1944), using the eggs of the gold- fish Carassius. They removed part of the uncleaved egg either by cutting it away with a knife or by pricking the future embryonic area and squeez- ing some of the cytoplasm out through the hole. They found that the earlier the operation was made after fertilisation, and the larger the part of the normal protoplasmic region remaining, the better was the differenti- ation. Fully normal embryos could be formed when the operation took place in about the first quarter of an hour after fertilisation and at least a half, or preferably more, of the protoplasmic region was left intact. In the abnormal and reduced embryos, mesodermal tissues sometimes differ- entiated rather well, but neural tissue does not appear to have been seen in the absence of accompanying mesoderm. The same authors also divided the early blastoderm into two parts, cut- ting along the cleavage planes in either the 2-, 4- or 8-cell stages. They found that each part might sometimes give rise to a complete and normal embryo. In other cases there was one normal embryo and one mass of cells with no histological differentiation ; and in some cases both portions gave this undifferentiated result. Two complete embryos can also be fused together, in stages up to the i6-cell stage. They sometimes produced complete, separate, twins, but occasionally one well-formed but oversized embryo to which it was clear that both eggs had contributed. All these results are exceedingly similar to those which have been found in the Amphibia, and might indicate a similar underlying mechanism, namely an organisation centre which becomes located in the plane of bilateral symmetry soon after fertihsation, which is essential for the development of the axial structures of the embryo, and which is capable of considerable EMBRYO FORMATION IN OTHER GROUPS OF VERTEBRATES 23I regulation, either when parts of it are removed or when two centres are fused together. But the critical evidence for such a centre is the fact that some blastomeres fail to give embryos. In view of the results of earher work on Fundulus, it is perhaps unsafe to lay much stress on such essenti- ally negative evidence. In the stages immediately following this, a phenomenon has been noted which has sometimes been claimed to be a special feature of teleost development with no parallels in other groups. Oppenheimer (1934/', 1936a) found that blastoderms of the minnow Fundulus, if removed from the yolk at the i6-cell stage or before and cultivated in salt solution, failed to gastrulate and developed only into featureless balls of cells. A rather similar result was reached for the goldfish by Tung, Chang and Tung (1945). They found that if eggs were divided latitudinally at the i-cell and 2-cell stage, blastoderms could not develop properly unless they remained connected with more than half the total quantity of yolk. From the 4-cell stage the requirement of yolk was less and from the 8-cell stage onwards completely isolated blastoderms could develop into embryos. Devillcrs (1947) found that the blastoderm of the trout was unable to develop in isolation from the yolk even when it had been removed as late as the blastula stage. On the other hand, in the pike Esox similarly isolated blastoderms continued developing quite well. The explanation of these facts is not clear. As a matter of fact a some- what similar phenomenon has been reported in the Amphibia. Vintem- berger (1936) showed that if, in the 8-cell stage of the frog, the four animal cells are isolated, they are usually unable to differentiate any axial organs although they contain part of the presumptive posterior region of the notochord rudiment. If however the four cells are placed on a base con- sisting of a mass of endoderm cells from the blastula, they differentiate very much better. There are several different ways in which these results and those in the teleosts could be interpreted. (i) It is a fairly general observation that early stages of embryos are more sensitive than older ones to the general injury produced by experi- mental operations. The increase in resistance usually continues at least until the main embryonic organs are laid dovm. It is noticeable, for instance, that it is easier to get good differentiation in tissue culture of chick blastoderms of primitive streak or later stages than of blastoderms taken from eggs in the first few hours of incubation. It may be that we are dealing merely with a general increasing 'tougliness' of the embryos, which varies from species to species. (2) Oppenheimer originally considered that the phenomena suggested the passage from the periblast into the blastoderm cells of some substance Q 232 PRINCIPLES OF EMBRYOLOGY of an organiser-like nature which was necessary for the initiation of gastru- lation (see 1947, where she is aheady somewhat cautious about this hypo- thesis) Tung, Chang and Tung considered their results supported this. The boundaries of the cells, particularly of the periblast but also of the blastoderm, are not very sharply marked in early cleavage stages of tele- osts, and it seems quite possible that the blastoderm contmues to mcor- porate further cytoplasmic material for some time after the cleavages have begun. (3) It may be, however, that what the yolk provides is not some org^- iser-hke substance but rather a relatively simple essential nutrient This possibility is supported by the fact that DeviUers (1949) found that blasto- derms of the trout, which are unable to differentiate in pure salt solution, will do so when glucose or similar substances are added to the medium. In a short note Trinkaus (1953) reports rather similar results with Fundulus. Only if the second of these possibihties is the full explanation ot the facts would we be confronted in the teleosts with a situation which differed radicaUy from that in the Amphibia (since it is improbable that a similar process is the explanation of Vintemberger's results). At present the matter must remain open. Experiments on the gastrula stage show that, in teleosts, the invaginat- ing chorda-mesoderm acts as an organiser m a manner extremely similar to that found in the Amphibia and in the chick. This was demonstrated almost simuhaneously by Oppenheimer for the perch and Funduus (1934a, h, i936h) and by Luther (193 5, 193?) for the trout Salmo (Fig. II. 5). The parallel with the amphibian and bird orgamsers is ex- tremely complete (see Review by Oppenheimer 194?)- For mstance there is a similar type of regional determination by the different parts ot the organisation centre, and evocation by dead tissues has been demon- strated It has been claimed (e.g. by Oppenheimer 1947) that the teleost ecto- derm has some tendency to differentiate neural tissue without the mter- vention of the mesodermal organiser. Oppenheimer tends to mterpret this in terms of an evocation by cytolysis, since in the abnormal embryos which gave evidence of such autonomous differentiation the cells were somewhat unhealthy. It appears however that the neural tissue was not completely without any accompanying mesoderm, but merely without accompanying differentiated chorda-mesoderm. The induction of neural tissue by mesoderm which does not include the chorda, and which may itself differentiate rather poorly, is well known both in the Amplnbia and the chick, and it appears unnecessary to postulate anything more tlian tliis to explain the phenomena in the teleosts. EMBRYO FORMATION IN OTHER GROUPS OF VERTEBRATES 233 It appears probable that there is a considerable difference of a quantita- tive, if not of a qualitative, character between the teleosts and the Amphi- bia in the extent of regulation which can go on in portions of the blastula and gastrula separated from the main centre of the organiser region. Luther (1936) divided the pre-gastrulation blastoderm of the trout^'into four equal quadrants, each of which was then isolated by grafting it into the yolk-sac epithehum of an older embryo. All four quarters were found capable of differentiating into all the main embryonic organs, although these were usually somewhat chaotically arranged owing to the Figure 11.5 In a an early gastrula of the trout is opened, the tongue of invaginating material cut away and grafted into the opposite side of the blastoderm; h surface view, showing the graft (dotted) ; c and d, stages in the developm^ni ot the host and mduced embryos. (From Luther 1935.) abnormal mechanical conditions. Soon after the beginning of gastrulation this equality between the four sectors disappeared. The sector diametri- caUy opposite the position in which the embryo will form soon loses its power to differentiate axial organs. There develops, in fact, a well-marked gradient, according to which the capacity for differentiation falls off from each side of the mam embryonic region. In Fundulus, Oppenheimer (1053) finds rather better differentiation of embryomc regions (three successful out of six) than of extra-embryonic germ-rmgs (five out of seventeen) but the figures are hardly sufficient to estaUish the existence of a gradient In the Amphibia at similar stages (i.e. blastula and early gastrula) the capacity of isolated ventral portions to regulate and form a complete embryo is, of course, very much less than that just described for the teleost 234 PRINCIPLES OF EMBRYOLOGY It is however not completely absent, as Dalcq and Huang (1948) have shown (p. 177). The teleost situation is, however, more comparable to that in birds at the time of endoderm formation, as described by Lutz (p. 183); there, again, the centre at which the embryo will form is not sharply 'distinguished from the rest, and far reaching regulation is still possible. Devillers (195 1) has studied the orientation in which embryos develop from portions of the trout blastoderm left in place on the egg or rotated in various ways. His results indicate that, in spite of the power of regula- tion which is spread throughout the whole blastoderm, the future embry- onic region has akeady some shght degree of dominance both in the blastula and still more the gastrula stages, and it tends to dictate the orientation of the embryo. This again is very similar to the situation m birds. 11111 c The well-known evolutionary changes affecting the head skeleton ot the fish have been discussed from the epigenetic point of view by Devillers (1950). 3. Reptiles Passing over the Amphibia, about which enough has already been said, we come in our survey of vertebrate types to the reptiles. They have been studied, recently, in the hght of our newer knowledge of other groups, particularly by Peter (1938) and Pasteels (1936-7, i94o). The main points which require notice concern the formation of the endoderm and the invagination of the mesoderm. The eggs are large and full of yolk; cleavage is partial; and we meet once more a blastodermal type of embryo. When the presumptive areas are mapped, it becomes clear that the make-up of the blastoderm is radically different from that of the teleosts. Instead of the presumptive endoderm and mesoderm lying round the margin of the blastoderm, they are located within it, the edge of the plate of cells being whoUy ectoder- mal. There is considerable variation within the group in the mode of endoderm formation. In the Algerian turtle Clemmys leprosa, Pasteels fmds that the blastoderm at the end of cleavage is single layered. At a point, which lies not at the edge of, but within, the blastodermic sheet, a groove appears and cells of the single layer become pushed inwards to form the endoderm, which thus owes its origin to a true invagination through a blastopore. In other species, such as the chameleon and certain hzards, the blastoderm never becomes single layered, and much, though perhaps not all, of the endoderm is formed in place by a delamination similar to that which was described above (p. I55) for the bird embryo. EMBRYO FORMATION IN OTHER GROUPS OF VERTEBRATES 235 After the blastoderm has become two layered, and endoderm formation is complete or nearly so, the invagination of mesoderm takes place through the same blastopore. The tissues move to the blastopore, sink down through it, and are pushed forward in the shape of a tube which extends towards the anterior. This used to be known as the 'archenteric canal', but since, according to the vital staining experiments of Pasteels, it contains no endoderm but is made up wholly of mesoderm, it should, he suggests, be called the 'chorda-mesodermal canal'. Its roof will eventually become notochord, its sides somites, while its floor migrates further later- ally to form the side-plates. At its anterior end the floor disappears at later stages, so that the canal leads from the blastoporal opening right through into the subgerminal cavity (Fig. 11. 6). Unfortunately, nothing whatever is known of the causal mechanisms of early reptilian development. The very slow differentiation of the eggs I I I I I I I I I I msid^^m^M^$^S^ FlGURE 11.6 Gastrulation in the turde Clemmys. A, map of presumptive areas, before the invagination of the endoderm (shading as in Figure 11.3, dashed vertical lines, extra-embryonic). B, movements of surface tissues tov^^ards the blasto- pore, from which the chorda-mesoderm canal extends. C, formation of endoderm from the blastopore at the beginning of gastrulation. D, the movements of tissues at a later stage through the blastopore into the chorda- mesoderm canal. (From data of Pasteels.) 236 PRINCIPLES OF EMBRYOLOGY makes them seem likely to be difficult experimental material, and no one has yet had the opportunity or the boldness to tackle them. For the sake of continuity, it is worth while repeating two ponits about the avian embryo in relation to what has just been said about the reptilian. In the first place, endoderm formation is probably by delammation, as m turtles, not by invagination from a definite blastopore. Secondly, while mesoderm invagination is beginning, streaming movements take place from the posterior towards the anterior; thus instead of the mesoderm beincr formed from a circumscribed blastopore which leads m to a long forwlrdly extending chorda-meso dermal canal, it originates from an elongated primitive streak, from the anterior end of which there juts forward only a short extension of invaginated material, namely the head process. 4. Mammals The major characteristic of the mammals (except for the most priniitive group) is the adaptation of their embryos to intra-uterine life. Evolution has in fact, been particularly active in the bringing about of changes m the early stages of development, and the group as a whole shows a very wide range of different conditions, which we shall only be able to sketch m very broad outline. In the lowest group, the prototherian mammals or monotremes, such as Echidna, the egg is still reptilian in general configuration, cleavage is partial, and a blastoderm is formed. Flynn and Hill (i939, 1942) fmd that endoderm formation takes place mainly by delammation, though there is also some movement of isolated cells out of the upper layer mto the deeper one. 1 \ 1 r r^-V, In the higher groups (marsupials and true mammals), the torm ot the ep-g is completely different (Review: Nelsen 1953). It is small, and con- tains little if any yolk. Cleavage is total. Endoderm and mesoderm forma- tion, however, occur by processes wHch are clearly modifications of those seen in the reptilian ancestors. The cleavage gives rise at first to a rather sohd mass of cells, but a cavity soon appears amongst them This is excentri- cally placed, so that the embryo assumes the shape of a hoUow sphere, or blastocyst, to the imier face of which a thicker cluster of ceUs adheres at one place (Fig. 11.7). This is the 'inner cell mass', and from it the mam embryo will be formed. The remainder of the sphere is extra-embryonic 'trophoblast', concerned with anchoring the embryo to the wall ot the uterus; it is probably to be compared with the outermost parts ot the reptihan and bird blastoderms, the area opaca. From the mner cell mass, the endoderm probably forms by simple delamination, though little is EMBRYO FORMATION IN OTHER GROUPS OF VERTEBRATES 237 known of this in detail and no vital marking experiments have yet been possible; there may well be some migration from the edges of the inner cell mass along the base of it towards the centre. The mesoderm is formed from the outer surface of the iimer cell masss. In this region the outermost layer of all, the 'enveloping layer' which is continuous with the main sphere of the blastocyst, either lifts away to leave a cavity (thus forming an amnion), or breaks dov^i and disappears. The face of the inner cell mass thus exposed has in general an oval form in plan view, and on it there appears an elongated thickening, wliich is the primitive streak. In some forms, such as the rabbit, it is extremely like Figure 11.7 Early development in a mammal. A, section of blastocyst, with itmer cell mass. B, later stage, the main part of the blastocyst becoming the tropho- blast (Tr), the lower layer of the inner cell mass becoming arranged as an epithelium, the endoderm. C, the iimer cell mass arranged as two epithelia, ectoderm and endoderm; above the latter the original outer layer has be- come elevated so as to form the amniotic cavity {R.in., 'Rauber's mem- brane'). that seen in birds; in others, such as the mole and in man, the streak is shorter and there is a greater development of a chorda-mesodermal canal such as that found in reptiles. The invagination of the mesoderm has never been followed in detail by vital staining, but a few cinematograph fdms of rabbit blastoderms growing in vitro have been taken by Waddington, and a convergence of lateral material towards the streak from both sides has been seen ; there is little doubt that the process is essentially similar to that of the bird and reptile. There are, of course, certain differences in detail. Thus in the rabbit the presumptive neural plate probably occupies a greater proportion of the embryonic area which corresponds to the area pellucida of the chick; and Waddington (1937) points out that the results of cultivation in vitro of posterior halves of rabbit embryonic areas 238 PRINCIPLES OF EMBRYOLOGY suggests that there is less longitudinal movement up and down the streak than in birds. 1 • r 1 The first few steps have been taken towards a causal analysis ot early mammalian development. Dalcq (1951^, 1952), and several of his pupils (cf Jones-Seaton 1950) are engaged in a detailed study of the differential staining and other properties of the cytoplasm of the oocyte and early developmental stages of the eggs of a number of species. Their most important results so far are, perhaps, the demonstration that there is some degree of bilateraHty in the structure of the oocyte and unfertihsed egg; and that the distinction between the embryonic and extra-embryomc regions arises gradually, and is not fixed at, for instance, the first cleavage division as has sometimes been suggested (Fig. 1 1. 8). . r^u- ■ The latter conclusion accords well with experimental work. This is, of course, technically very difficuk in mammals. It is first necessary to operate on the young stages, to remove the eggs from the mother, then to divest them of the tough jelly which surrounds them or to operate through it, and fmally to return them to some situation m which they will continue their development. Rodent eggs, which are easy to obtain, seem very sensitive to the removal of their membranes, and are ditticult to keep alive in any situation except the uterus, although they can be re- transplanted back into another uterus with fair success (cf Nicholas 1947, WiUett 1953). The rabbit egg is in general tougher; the embryonic area at the primitive streak stage can be cultured in vitro for a time long enough for a fair amount of differentiation to occur. It is worth notmg (cf p. 231) that the older the embryo, the better it stands the conditions of artificial cultivation (Waddingtoni937). ...11 j A httle work has been done on the early cleavage stages. Nicholas and Hall (1942) succeeded in getting some development (up to just before the time the embryo makes its appearance) of one of the first two blastomeres, the other having been destroyed or removed. Pincus (1936) got rather similar resuks with the rabbk. Recently Seidel (1952^ had been much more successful. One of the first two blastomeres of a rabbit egg was kiUed by pricking with a glass needle, without removing the jeUy layer, ihe eg/was then mjected mto the Fallopian tube of another rabbit at a suit- able stage in the reproductive cycle, and fully norm.al young were born. The evidence that one blastomere was really killed seems quite con vmcmg, and the operated egg differed genetically (in colour) from the host, so that there is no doubt that the young animal was derived from it and not from an unkijured egg of the foster-mother. Only two such ammals have so far been described; one was fully normal, but the other showed defects on one side, rather similar to those which are found m newts eggs from EMBRYO FORMATION IN OTHER GROUPS OF VERTEBRATES 239 half blastomeres when the first cleavage furrow does not divide the grey crescent region into equal parts. One may conclude that the rabbit egg (and presumably that of other mammals) is certainly not of the mosaic type, but may contain a localised organisation centre comparable to the amphibian grey crescent; this would probably be related to the bilateral structures described by Dalcq. (For experiments involving the alteration of chromosome numbers, induction of polyploidy etc. in mammals, see Beatty 1955.) Figure 11.8 Three stages in the early development of the rat. Upper row, fertiHsed egg with pronuclei; middle row, four-cell; lower row, young blastocyst with inner cell mass. The eggs in the left column have been stained to exhibit alkaline phosphatase; those in the centre column RNA; and those in the right column mucopolysaccharides. (After original drawings of Dalcq.) A few experiments have also been made at the primitive streak stage of rabbits although the material is technically very difficult to handle owing to its stickiness, transparency and resistance to cutting. Wadding- ton (1937), failed to get any inductions by fragments of primitive streak transplanted between the epiblast and hypoblast in the manner used in the chick, but the material was certainly not extensive enough for this nega- tive result to have much importance. On the other hand, the competence 240 PRINCIPLES OF EMBRYOLOGY of the rabbit epiblast to react to neuralising stimuli was demonstrated by cases in which such an effect was produced by grafts of chick primitive streak (Fig. 11.9); and rabbit primitive streak was also able to induce when grafted into the chick. It is highly probable, therefore, that the mammalian primitive streak is an organisation centre with functions similar to those of the same structure in the bird embryo. It was also showai that development of the posterior region is possible in the abseace of the anterior end of the streak (Hensen's node), which some authors had considered to be an essential focus of embryo formation ; again a result which parallels that in the bird. Toro (1939) has reported the induction of neural tissue in the rat by grafts placed in the amniotic cavity, but his Figure 11.9 A small piece of chick primitive streak was grafted under the ectoderm of a rabbit embryo of the streak stage, which was then cultured in vitro. The graft has formed some neural tissue, and has induced a neural plate in the rabbit host. (After Waddington 1934.) evidence, as published, is not very convincing, and it would be unexpected to find that tissues lying freely in such a cavity, in contact with the outer side of the ectoderm, could successfully induce; it seems more likely that he was dealing with embryos which were distorted as a result of the operations. The results of direct operative experiments on mammal embryos have been supplemented by evidence of quite a different kind, which comes from an analysis by methods which have as yet been little used in other groups of animals, except the insects. A fairly considerable number of hereditary factors or genes are known which cause abnormahties in early developmental stages of mammals ; perhaps their comparative frequency in this group is connected with the extremely rapid and radical changes which have led to the evolution of the group. The effects of these genes are often somewhat variable, and by a study of the variations it is some- times possible to decide that one particular aspect of the abnormality is primary, and the remainder secondary consequences; from such argu- ments some insight into the causal sequences of epigenesis can be attained. EMBRYO FORMATION IN OTHER GROUPS OF VERTEBRATES 24I Dunn (1941) and in particular Gluecksolin-Schoenheimer (1949, 1953) working on the mouse, have made very important contributions in this way. The first genes from which important embryological consequences could be deduced had their most obvious effects on the tail. The Brachyury gene T is a dominant, the heterozygous mice having short tails. The Tj T homozygotes die before birth and in the embryos the whole posterior region of the body is missing (Chesley 1935). Detailed investigation showed that no notochord (or almost none) is ever formed in these homo- zygous embryos, and Chesley suggested that this was the primary action, the effect on other structures such as the nerve-cord being the result of secondary reactions of an inductive nature. This was confirmed, and the evidence made more convincing, when Gluecksohn-Schoenheimer in- vestigated embryos of animals wliich were heterozygous both for T and for one or other of the genes t^ and f^. These embryos have normal- looking tails till an age of about eleven days, after which the tail becomes constricted at its base and degenerates, the young being quite tailless when bom. Histological examination showed that even in the apparently nor- mal tails of early stages, no notochord is present, although the neural tube, somites and tailgut are formed as usual. It seems then that the later degen- eration is a consequence of the lack of the notochord, and some 'inductive' relation is indicated. It should be noted, however, that the relation is not quite that of evocation ; even in the absence of the notochord, the neural tube is formed, presumably induced by the remainder of the mesoderm, but it is unable to persist. There is no exact parallel to this in other verte- brates, since the operative removal of the notochord, e.g. in Amphibia, does not lead to the regression of the neural tube, but only to a failure of its normal elongation and an alteration of its usual cross-sectional shape. The homozygous t^/t^ embryos show more profound abnormalities. The whole endoderm tends to hft away from the rest of the inner cell mass some time before the beginnings of embryo formation are visible. The cells of the mass remain alive for some time, and some growth takes place, but there is no organisation of a primitive streak and no mesoderm appears; a day or two after the onset of the condition, the embryos die and are resorbed. Gluecksohn-Schoenheimer suggests that this may indi- cate that in the mammals, as in the birds, the endoderm may play an essential role in inducing the formation of mesoderm. The same author has described a still more interesting gene, known as Kink. The heterozygotes show various spinal and tail abnormalities, and the homozygotes die at about nine days after fertilisation. Before death, a considerable amount of development has occurred, and the embryos 242 PRINCIPLES OF EMBRYOLOGY exhibit a most remarkable range of conditions, most of which can be considered as twinnings or duphcations of various kinds, ranging from almost complete and separate twins to doublings of the main axis, of the heart, the allantois, etc. There are also many examples of single more or less complete embryos accompanied by ball-shaped lumps of unorganised tissue. Gluecksohn-Schoenheimer compares the structures found to those which result from the partial or complete separation of the first two blastomeres in Amphibia. If this comparison is accepted, and it seems quite convincing, one would have to conclude, firstly that the mammal egg at an early stage is capable of profound regulation (a point already clear from the occurrence of identical twins and directly confirmed by the evidence from blastomere separation mentioned above [p. 238]); and secondly that it possesses an organisation centre which is, or becomes, locahsed as does the grey crescent in the Amphibia or the posterior part of the endoderm in birds. The most plausible explanation of the action of Kink is that it interferes with the gradual regionalisation within the egg, by which the organisation centre becomes focused on to the dorsal side. Unfortunately the evidence does not help us to decide at what stage in development this takes place in mammals. In the armadillo, in which four identical twins are normally produced from each egg, the initiation of the four rudiments appears to occur rather late, in the blastocyst stage (Patterson 191 3), and although one would now like to see the newly fertihsed eggs of this form re-investigated by the methods of Dalcq, it is perhaps likely that the mammals are like teleosts in that their dorsal plane is not finally fixed till a relatively late stage. It may be mentioned that all the mouse genes just mentioned (T, t°, t^ and Kink) are closely linked in the same chromosome; they may all be chromosome aberrations rather than true point mutations. Many other genes with generally similar effects (mostly not yet analysed embryologic- ally) are known in the same chromosomal region. The causes for the association of this part of the chromosome with the primary organiser phenomena is quite unknown and present a very intriguing problem. Gluecksohn-Schoenheimer very tentatively suggests that possibly the spatial pattern of the genes in the nucleus may have some connection with the formation of the patterns of the early embryonic fields. 5. Comparative geography of the presumptive areas In the past, attempts to compare the processes of gastrulation in the different classes of vertebrates have been made in terms of concepts which were derived from the static shapes which particular embryos may assume at certain points in their development. For instance, one tried to EMBRYO FORMATION IN OTHER GROUPS OF VERTEBRATES 243 homologise the structure of the blastoderm of the bird before endoderm formation begins with the hollow sphere formed by the amphibian egg at the end of cleavage; or one enquired how the chorda-mesodermal canal in reptiles is related to the amphibian blastopore. Nowadays we regard gastrulation as a co-ordinated series of foldings and movements by which the various groups of cells which result from cleavage are arranged into the three fundamental layers of the ectoderm, mesoderm, endoderm. "What ought to be compared is the complete set of movements in one form with the complete set in some other form, rather than any particular instantaneous configurations which may be taken up during the process. The most manageable way of summarising the whole gastrulation of a particular group is provided by the map of presumptive areas at the late blastula stage, together with an indication of the directions in which these various areas will move. When one looks at the maps of presumptive areas which have been described earher, it is fairly easy to arrange them into a scheme in which they are brought into natural relations with one another. It is clear that such a scheme should start from a type of egg which is fairly small and has total cleavage, since the blastodermic forms of development must be secondary derivatives. Of the totally cleaving eggs, the most primitive fish (cyclostomes) and the Amphibia have maps which greatly resemble one another. The blastopore is placed low down on the vegetative side within an area of endoderm. Above this is a ring of presumptive meso- derm, wliich, certainly in the Amphibia and probably in the cyclostomes, extends right round the egg from the dorsal to the ventral side. Within this ring the presumptive axial mesoderm (notochord and somites) is con- centrated somewhat towards the dorsal side, but extends much further laterally in the blastula than it will do after gastrulation is completed. The upper part of the egg is occupied by a cap of presumptive ectoderm, of which the region near the dorsal plane will become neural plate and the remainder epidermis. A very similar pattern is found in the protocordates (ascidians and Amphioxus), and we are probably safe in taking this as the basic type from which the others should be derived (Fig. ii.io). There are two other main types to be fitted in. Between the cyclo- stomes and the Amphibia in the evolutionary sequence come the cartilaginous and bony fishes. These have a blastodermic type of develop- ment, and a characteristic feature of their presumptive map is that the margin of the blastoderm is made of presumptive endoderm or possibly mesoderm; the whole ectodermal area lies well inside the margin. The other groups — reptiles, birds and mammals — are evolutionarily more advanced than the Amphibia. Their embryos also develop from a 244 PRINCIPLES OF EMBRYOLOGY blastoderm but the presumptive map differs radically from that of the fish in that the edge of the blastoderm is in this case constituted of ectoderm. The presumptive mesoderm lies inside the margin and when the endoderm occurs on the surface, as it does in certain reptiles, it is the most centrally placed area of all. As between reptiles, birds and mammals, there are obviously great similarities, such differences as are found being dependent on the extent to which the endoderm is formed by delamination or is AMPHIBIA blastopore TELEOSTS REPTILES AND BIRDS Figure ii.io Maps of presumptive areas. The large circles in the teleost and reptile maps represent yoUc; other conventions as before. In the amphibian map, A represents the point at which the yolk would have to be inserted to give the teleost map, and B that required to give the reptile-bird map. (From Wad- dington 1952.) originally at the surface, and on the degree to which the site of mesoderm invagination is drawn out into a primitive streak or concentrated into a chorda mesodermal canal. The movements in the eggs of all the groups are rather similar in general type. There are movements of expansion in all directions, affecting parti- cularly the ectoderm. There is a 'dorsal convergence', in which tissues be- come narrower from side to side and simultaneously elongated, and do this the more intensely the nearer they lie to the dorsal plane. Again, there are tendencies for invagination, that is, for certain tissues to plunge inwards from the surface and continue their movements at a lower level. Finally, EMBRYO FORMATION IN OTHER GROUPS OF VERTEBRATES 245 in the blastodermal types, we fmd delaniiiiation, that is, a process by which ail original single thick layer of tissue becomes rearranged into two thin- ner separate layers. Until we know more of the mechanical causation of the movements, it is not possible to make any further meaningful com- parisons between them. The main aspect of the comparative scheme which one would like to understand more fuUy is the relation between the two sorts of blasto- dermal development. Two main ways of regarding this have been pro- posed. Dalcq (1938) and Pasteels (1940), who were the first to discuss the matter at all fully, were content to accept an irreconcilable duahty within the evolutionary system of the vertebrates. According to their scheme, the teleost map should be regarded as derived from that of the Amphibia by the insertion of a large mass of yolk near the vegetative pole, while the bird-reptile map is derived by inserting the mass of yolk somewhere within the area of presumptive epidermis (at point B in Fig. 11 . 10). Tliis is the simplest and most straightforward scheme, but the idea of such drastic quahtative differences of egg structure within the vertebrates is not entirely satisfactory, Waddington (1952&) has suggested that one ought to take into account not only the disposition of the presumptive areas on the egg surface but also their arrangement in depth. The blastodermal types might be derived from the ampliibian arrangement, not by opening the latter at some point and spreading it out so that it can sit as a cap on top of a large mass of yolk which is inserted in the hole, but rather by imagining that the amplii- bian blastula becomes yolk-free, and then that the whole sphere is squashed down on to the surface of the separated yolk mass (Fig. 11. 11). If, when the spherical blastula is distorted in this way, the line along which it folds (the 'primitive edge') lies whoUy within the presumptive mesoderm, then the blastoderm will have a mesodermal margin and the whole of the endoderm will already be beneath the surface before gastrulation begins. If the primitive edge on the dorsal side lies a little lower, within the eiido- dernial region, some of the dorsal margin of the blastoderm will be endo- dernial. According to the exact position of the line, one can easily arrive at the conditions seen in the Selachia or teleosts. Again, if the primitive edge lies liigher on the amphibian map, it will, particularly on the ventral side, cut into the presumptive epidermis and the blastoderm will have an epidermal margin in that region. In order to explain the fact that the whole of the blastodermal margin in birds is ectodermal, we should have to postulate an expansion of the presumptive epidermal area in addition to the flattening of the spherical blastula. This makes the scheme more complex than that of Dalcq and 246 PRINCIPLES OF EMBRYOLOGY Pasteels. Its main merit is that it gives an example of one way in which it is possible to envisage both the fish and bird blastoderms as two modahties of one general process ; and further, it draws attention to the need to con- sider the disposition of the presumptive areas in depth as well as on the surface. It is most desirable that we should find out more about the loca- tion of the presumptive areas in the extremely yolky types of amphibian eggs, particularly those of the Gynmophiona. In these, much of the en do- derm seems to be beneath the surface before gastrulation begins and to become distinct by a process of delamination, which would seem to accord better with Waddington's scheme than with the Dalcq-Pasteels' one. Gastrulation in these eggs has, however, not been studied since Brauer's work in 1897. ventral ventral dorsal dorsal O o o o o o 000 Figure ii.ii A, the basic vertebrate map, with a line of folding ('the primitive edge') indicated by a broken line. B, the blastula squashed dowoa on to a mass of yolk, having folded along the primitive edge. If such a deformation is to produce the bird map, there would also have to be an expansion of the epi- dermal area. (From Waddington 1952.) 6. Comparative causal embryology of vertebrates The various types of vertebrate eggs, different in many respects and yet all anatomically related in the ways we have just discussed, provide a wonderful opportunity for studies on the kind of alterations in epigenetic mechanisms which evolution can bring about. There is no doubt that one ought eventually to envisage evolution not as a process wliich merely causes modifications in the structure of adult animals, but rather as one by which the causal sequences of development have been changed. These changes have affected even the basic mechanisms which operate in the early stages of development. Dalcq [igsib) has referred to mutations which affect these stages as 'onto-mutations'. Little is known in detail about them from the genetical side, though many of the factors which are labelled (and usually dismissed) as 'female-steriles' probably fall into tliis category EMBRYO FORMATION IN OTHER GROUPS OF VERTEBRATES 247 (cf. p. 135). The comparative study of the epigenetic systems in different groups of vertebrates gives an opportunity to see the kinds of effects which such gene changes produce. The first noteworthy epigenetic event in the development of vertebrates is the fixing of the plane of bilateral symmetry. In the Amphibia, as we have seen, the future organisation centre normally becomes located soon after fertilisation and is sometimes visible as the grey crescent. In the other primitive non-yolky type of vertebrate egg, that of the cyclostomes, Montalenti and Maccagno claim that the situation is very similar. In the protocordates (ascidians and Amphioxus) the dorso-ventral plane is also fixed definitely at an early stage. In both the ascidians and amphibians there is indeed some evidence that the dorso-ventral plane is at least fore- shadowed before fertilisation occurs. In the latter the position of the plane is at first labile and only gradually becomes definitely deter- mined. By the time cleavage is completed and the blastula stage reached, the dorsal plane is firmly determined and it is difficult to cause a new dorsal region to appear elsewhere when the egg is divided in two. Such powers of regulation are, however, not completely extinguished until consider- ably later (p. 177). The results obtained by Bytinski-Salz on the cyclostomes suggest that the same is true in that group. In the blastoderms of both the teleosts and the birds the situation seems to be rather different. We have some evidence (Vakaet 1953) that the growing oocyte of the teleost has a bilateral structure, but in the develop- ing embryos the position of the dorso-ventral plane is quite easily alter- able until a late stage of the blastula, as is shown by the results of Luther in teleosts and Lutz in birds. It seems probable that the original bilaterahty in the oocyte is swamped by the great flood of yolk which is laid down, and that a bilateral structure is only gradually and slowly re-established. It is true that Tung and Tung (1944) argued that, in the goldfish, an organ- iser-like region beomes located on one side of the egg shortly after fertili- sation, and thus endows it with a bilateral symmetry, but it is doubtful if their experimental material was adequate to sustain this conclusion (p. 230). It seems likely therefore that we have to accept a difference in timing as between the holoblastic and blastodermal eggs, the dorso- ventral plane being determined much later in the latter. In the Ampliibia the agent wliich determines the dorso-ventral plane was the inner yolk mass. In the teleosts it seems to be the periblast. It is difficult to say how far these two can be considered as in any way com- parable, either in their anatomical derivation or their mode of action. In both groups what is determined is, in the first place, the invagination of endoderm; and hard on the heels of this comes the mesoderm, which 248 PRINCIPLES OF EMBRYOLOGY proceeds to be invaginated arouiid the same focus. In birds we have no direct evidence as to the mechanisms v^liich determine the posterior end of the cndoderm, v^hich is the region that takes the lead as soon as the dorso-ventral plane is fixed. In tliis form, however, another stage is inter- polated into the normal sequence, since there is something of a pause be- tween cndoderm formation and the start of invagination of mesoderm at the primitive streak, and we find that the cndoderm exerts a causal influ- ence wliich determines the site of mesoderm invagination. The apparently straightforward, follow-my-leader behaviour of mesoderm towards cndo- derm m the lower groups has, therefore, been expanded in the birds into a cause-effect sequence. We still know too little about the epigenetics of mammals to fit them with any confidence into the scheme. There is evidence (Dalcq, p. 238) that the oocyte and newly fertilised egg have some degree of bilaterality of structure. Seidel, as a result of his separation of blastomeres, argued that there is a localised organisation centre. This result, however, was based on only two sets of operations and is exceedingly tentative. On the other hand, the evidence suggests that the formation of identical twins may occur at much later stages; and on the whole it seems probable that in mammals, as in birds, the final determination of the dorso-ventral plane occurs rather late. It seems probable that, in all the vertebrates without exception, deter- mination of the dorso-ventral plane fixes, in the first place, the location of the cndoderm and, secondly, that of the mesoderm, but that the ecto- derm remains quite indifferent till it is acted upon by an organising influ- ence coming from the mesoderm. Grafting experiments have given direct proof of the inducing power of the presumptive mesoderm in cyclostomes, teleosts, Ampliibia and birds and the demonstration is only slightly less clear-cut in mammals. In all groups there is evidence that at the time the mesoderm starts to be invaginated the fate of particular regions of it can still easily be altered, but that, as invagination proceeds, it is affected by a process of regionahsation so that different areas become determined, not only as to the tissue into which they will develop (as notochord, somites, nephros, etc.), but also as to their position on the anterior-posterior axis (brain, spinal cord, tail, etc.). There is some not very compclhng evidence (p. 187) that in birds the presumptive forebrain has a tendency to develop into neural tissue independently of any induction by mesoderm. If this is so it may be connected with the fact that, in this group, the determina- tion of the site of mesoderm formation (the primitive streak) is a com- paratively long-drawn-out process caused by the cndoderm. In other groups the development of neural tissue seems to be completely dependent EMBRYO FORMATION IN OTHER GROUPS OF VERTEBRATES 249 on induction, and the whole responsibility for the appearance of the main embryonic axis can be attributed to this process and to the self-individua- tion (i.e. regionaUsation) of the mesoderm. It seems therefore that all the groups of vertebrates employ essentially similar epigenetic mechanisms. The main changes which have occurred within the group appear to be: (i) The swamping of the initial structure of the oocyte by the enormous quantities of yolk laid down in fish and birds, and a consequent postponement of the time at which the plane of bilateral symmetry is fmally determined, and (2) the interpolation of a new causal relationship between endoderm and mesoderm in birds (and possibly in reptiles and mammals) in. consequence of the difference in the time of invagination of the two layers. SUGGESTED READING Needham 1942, pp. 320-30; Dalcq 1938, pp. 7-58. For comparative gastrulation, Pasteels 1940, Waddington 1952^ or I952fl, pp. 39-50. For teleosts, Oppenheimer 1947; for mammals Gluecksohn-Schoenheimer 1949, Dalcq 1951a. CHAPTER Xn ORGAN DEVELOPMENT IN VERTEBRATES IT is clearly impossible, iii a book of this size, to do more than sketch in very broad outline the extremely complex processes by which the details of the adult anatomy come into being. There are, moreover, already many excellent accounts of them; for more extended comparative treatments, Dalcq and Gerard's revision of Brachet (1935), and particularly Nelsen's recent work (1953) can be recommended. In the present chapter, an attempt has been made to single out those aspects of organ development in which new advances have recently been made, or which are particularly important in connection with various general principles of development; and two organs, the hmbs and the kidneys, which provide very interesting illustrations of a number of points, have been dealt with in rather fuller detail. I. The general form of the embryo At the end of gastrulation, the amphibian embryo — often known at this stage as a neurula — is still a solid, approximately spherical object. The neural system runs along its dorsal meridian, at first in the form of an open neural plate, which however rapidly closes up into a neural groove and fmally a tube. It also stretches considerably in the anterior-posterior direc- tion, continuing that process of elongation which we have seen to be characteristic of the dorsal region through gastrulation. At an early stage in this stretching, the head and tail curl round the opposite ends of the egg, which has now become somewhat ovoid, so that the embryo seems to be on the point of biting its tail ; but as the elongation becomes still greater, particularly in the more posterior regions, the dorsal axis straigh- tens out again and the creature begins to assume the form of a tadpole, with a long thin tail stretching out behind its body. During all this time, there has been a tubular gut, formed directly by the invaginating meso- derm and endoderm, and opening out to the exterior through the blasto- pore, which lies at its posterior end in the region where the definitive anus will eventually be. At first the tube has a floor and walls of endoderm and a roof of mesoderm, but the lateral edges of the endoderm soon grow in from each side to meet in the midline and from then on the gut-tube is hned entirely by endoderm; we shall see that this situation is not so easily brought about in the flat blastoderm of the birds. The wall of the 250 ORGAN DEVELOPMENT IN VERTEBRATES 25I amphibian gut is at first rather thin on the dorsal side, but very thick on the ventral, where the cells are still swollen by the large stores of yolk. From the exterior, it appears as though little is happening on the ventral side while the tail is growing out, but beneath the skin the first blood cells are forming, and the mesoderm is beginning to form a pulsating tube, the rudiment of the heart. The chick embryo, when it reaches the neural plate stage, is not, to speak crudely, a solid lump like the amphibian, but is instead a flat circular plate, on which the embryo proper is linear in form, the remainder of the plate being no part of the final body but concerned only with the absorp- tion and digestion of the yolk and with the respiratory exchanges between the embryonic blood and the outside air. One of the most striking features of the embryo proper is the very great difference in the stage of develop- ment reached by the anterior and posterior parts. In the Amphibia the whole neural plate folds up more or less simultaneously to form a neural tube, which stretches from the anterior right to the posterior end, where there is only a small region of *tail-bud' at which new additions of neural tissue and mesoderm continue to be formed. In the cliick on the other hand, at a time when the anterior neural plate has folded up into a com- pletely closed tube in the head region, it is stiU only a shallow groove at the level of about the tenth somite, while posterior to this there is still a remnant of the primitive streak at which mesoderm is being formed and the primary organiser is active. Thus although differentiation progresses roughly from anterior to posterior in both forms, the differences are much more marked in the chick than in the frog or newt. Fairly soon after the closure of the neural tube in the anterior region of the chick, one can see the beginning of a process by which the embryo proper eventually becomes separated from the remainder of the blasto- derm. Along a crescent-shaped line in front of the head, the blastoderm is tucked downwards and backwards under the neural tube. The fold so formed is known as the head fold. As it gets deeper and is pushed further backwards, the head region is gradually left isolated on a projection above it. At a considerably later stage, when the embryo has some thirty or more pairs of somites, a similar fold (the tail fold) cuts under the posterior end of the embryo ; and eventually these two folds each progress so far to- wards the centre of the body that the whole embryo is left attached to the non-embryonic blastoderm only by a narrow stalk (Fig. 12. i). As the blastoderm is tucked downwards in the head fold, a pocket lined with endoderm appears on the under side of the head. This is the first rudiment of the gut to appear, and is known as the foregut, to distinguish it from the hindgut which appears later in comiection with the tail fold. 252 PRINCIPLES OF EMBRYOLOGY Bellairs (1953, 1954) has recently studied its development in detail, with the aid of vital stained marks. The foregut opens posteriorly on to the space betw^een the blastoderm and the yolk, and its posterior edge is a clearly defmed landmark in early chick embryos. As the head fold is pushed further and further backwards under the embryo, so the opening of the foregut moves posteriorly; in fact it does so even faster than the head fold progresses, and it is probable that its movement is not as simply dependent on that of the head fold as appearances might at first suggest (Waddington 1952, p. 149). After the opening to the foregut has pro- gressed some distance towards the posterior, the mesoderm in the edge of the fold develops into a hollow tube, which at first extends some dis- tance along each side of the crescent-shaped opening. This tube is the Foregut Figure 12.1 Hind^ut Diagrammatic longitudinal section through a chick embryo, to show the relation of the embryo to the yolk. rudiment of the heart. It is really a double structure, since the mesoderm which forms the tube on the left side originally lay well out at the left of the blastoderm, and a considerable distance away from that which forms the tube on the right side; it is only the backward progress of the edge of the foregut, which takes place by a sort of zip-fastener process along the midline, which has brought them together. This obvious doubleness in the early rudiment of the heart is one of the points in which the cliick embryo seems to differ most radically from the amphibian type. Even so, the two systems are not completely different. We can relate them by the imagining what would happen if the central part of the chick blastoderm were wrapped round a spherical core of yolky endoderm; if the two presumptive heart regions were at the edges of this part of the blastoderm, they would come into contact on the ventral ORGAN DEVELOPMENT IN VERTEBRATES 253 side of the endoderm and no further folding would be needed to bring them together; we should in fact have arrived at the amphibian condition. The reverse of this can actually be achieved experimentally. Nieuwkoop (1946) has slit an amphibian neurula up the ventral midline, removed the endoderm and flattened out the mesoderm and ectoderm on a plate of stretched silk, so that it is forced into a sort of 'blastodermic' configura- tion. It then develops two heart rudiments, one on each side, which fail to fuse so as to form a single heart, since the embryo is not pro- vided with the foregut-forming mechanism which brings this about in the chick. In the stage immediately after its formation, the heart of the chick embryo continues to have an important influence on the general form of the body. In the first place, it becomes extremely large. This is necessary because it has to pump blood not only through the embryo itself, but for very much longer distances through the blood vessels which run into the non-embryonic blastoderm. It becomes so disproportionate to the body proper that it bulges out to one side. At the same time, the whole body rotates, so as to lie with its left side down against the yolk; and the head also arches round so that the anterior end of the brain is bent down towards the chest, with the heart protruding between them. It is the development of the heart and its associated structures which is the cause of most of these foldings and rotations, and if it is removed, the main part of the neural system remains straight, only the anterior tip of the brain bending down- wards. 2. The development of the head In both the amphibian and the bird, the neural plate is wider in the anterior than in the posterior. This is very clear in the newt, in which the whole plate is marked out on the surface of the egg at the time the blasto- pore is closing; it is definitely pear-shaped. As the edges of this area fold together to form the neural tube, the future brain, developing from the anterior end, has from the beginning a wider lumen than the hinder parts of the nerve cord. At first the tube remains open at its anterior tip, the small hole connecting the interior of the tube with the outside being known as the anterior neuropore; it closes gradually during the later development of the brain. The comparatively slight initial difference in the width of the brain and of the main nerve tube soon becomes greatly increased by the appearance of swellings in the former. At first there are three, which form the primary brain vesicles which give rise to the forebrain, midbrain and hindbrain. Fairly soon the first and third swellings each become differentiated into 254 PRINCIPLES OF EMBRYOLOGY two, SO that the brain comes to consist of five vesicles, whose names are shown in Fig. 12.2. The division of the forebrain into two starts early, but progresses slowly. Even before it has properly started, the formation of the eyes begins. They are developed from the region which will eventually form part of the second forebrain vesicle (the diencephalon), and are originally merely exaggerated lateral swellings of the brain tube. These bulge out through FOREBRAIN Tehnciphalon Diencephalon MIDBRAIN — lAesenctphalon HINDBRAIN Metencepkalo^ Muencephalon Masai placode Lens placode acodt Figure 12.2 Diagram of the various regions of the brain, the eye-cups, and the nasal, lens and ear placodes. the mesenchyme which lies between the brain and the ectoderm (Fig. i.i, p. 7), and come up against the latter. At this stage, two things happen. The first is the formation of the 'optic cup' ; the swelling be- gins to be transformed into a roughly mushroom-shaped structure, with a rather narrow stalk leading up to the brain while at the other end the part wliich comes into contact with the ectoderm is folded inwards rather as one pushes in the foot of a sock before puttiag it on. The cavity thus formed is the eye-cup, and is approximately circular except for a groove, the choroid fissure, on the ventral side. The layer of tissue wliich lines the cavity of the cup becomes the light-sensitive retina, and the outer layer or ORGAN DEVELOPMENT IN VERTEBRATES 255 tapetum develops into part of the pigmented and protective coverings of the eye. The second ingredient in the developing eye is the lens. This originates from an infolding of ectoderm at the point where the optic swelling touches it. Its inception normally depends on a process of induction. Some time before Spemann discovered that the appearance of the main axis of the embryo depends on induction by the primary organisation centre, he had found that the eye-cups of newt embryos, transplanted so as to come in contact with the ectoderm of the belly, caused the appearance there of an induced lens (Reviews: Mangold 193 1, Spemann 1938, Need- ham 1942). The experiment reveals a classical example of a secondary organiser, that is, one which is effective after the primary organiser activity is com- pleted. Very many of these operate during the period in which the earliest organ rudiments are appearing (Fig. 12.3). In fact it is probable that in the Ear capsule I Forebrain] | Midbrain] \|Hindbrain| "Lumen of Kut Neural tube T Figure 12.3 Diagram of secondary organisers in the newt. (After Hollfreter.) vertebrates every rudiment owes its origin to a secondary organiser or to a complex of organising influences ; they are found in all the vertebrate classes which have been studied. For instance, lens induction by the eye- cup has been demonstrated by Waddington, van Deth and others in the chick (Review: Waddington 1952^7), And to give further examples, two other organs which require notice at this point also depend on secondary organiser complexes. The nervous component of the nasal organ arises as two ectodermal thickenings or placodes near the anterior end of the forebrain; and it can be shown by transplantation experiments that the forebrain induces them. Again, the ears originate near the hindbrain from similar lens-like ectodermal thickenings, around which, after they have been folded inwards, the mesodermal structures are later formed. Experi- ments have shown that in this case the inducing system is complex; the 256 PRINCIPLES OF EMBRYOLOGY hindbrain plays an important part in calling forth the thickenings, but so does the local mesoderm, and a fully normal ear can only develop when both components induce together in a harmonious way. There are in reality whole sequences of 'secondary' organisers, acting one after the other; perhaps they should be classed as secondary, tertiary, quaternary organisers and so on, but it would be difficult to do this in any very sensible way. For example, after the eye-cup has induced the lens, the whole complex then induces the skin lying above it to become trans- parent and to differentiate into the cornea, and the mesoderm which clothes it to become the sclerotic coats of the eyeball. Again the original ectodermal placode of the ear induces the mesoderm to form the other parts of the ear structure. Within its ectodermal covering, the head of course contains not only the brain but also an infilling of mesoderm and some endoderm. The latter forms the pharynx, or anterior part of the gut, and will be discussed below in connection with the latter. It remains here to say something about the mesoderm. This tissue has a double origin. The greater part of it is formed by invagination through the blastopore or primitive streak, and this is the part to which we have so far paid most attention ; but some mesoderm is also formed from cells which follow quite a different path. When the two neural folds finally come together and fuse to form the neural tube, some of the cells at the two fusing edges break loose and move down between the tube and the overlying ectoderm. These have been given a variety of names by various authors ; sometimes they are alluded to as 'mesectoderm', a word which is also used for the epiblast of a blastoderm before the mesoderm has invaginated and thus become separ- ated from the ectoderm; a somewhat better name is 'ecto-mesoderm', which is not so ambiguous, but probably it is simplest and best to speak of tliis second contribution to the mesoderm as the 'neural crest material', a phrase which clearly describes what is meant (Fig. 12.7, p. 266). In most of the trunk, the neural crest material is scanty compared with the whole bulk of the mesoderm; it eventually forms the pigmented cells of the skin and contributes to the spinal ganglia. In the head it plays a much more important part, and forms large masses of tissue, which develop not only into some of the cranial nerves but also give rise to many parts of the cartilaginous skeleton (Horstadius and Selman 1942, de Beer 1947)- 3. The gut: anterior portion In the Amphibia, it looks at first sight as though the formation of the gut is extremely simple, since a complete closed tube is formed during the ORGAN DEVELOPMENT IN VERTEBRATES 257 process of gastrulation (but see p. 261). At first the endoderm forms only a trough, which is covered dorsally by the mesodermal layer of which the notochord is a part, but fairly soon the edges of the trough grow round to meet under the notochord, so as to form a closed tube made wholly of endoderm. The anterior part of this tube reaches forward under some of the head structures which have just been described. At its tip, the tube is at first closed, and in this region there is no mesoderm lying between it and the ectoderm. In the mesoderm-free area, the gut first fuses with the ectoderm, and then both layers break down, so that an opening appears leading from the exterior into the lumen of the gut; this is the rudiment of the mouth. Just posterior to this, the gut becomes swollen into a large cavity, which is known as the pharynx. The wall of this becomes thrown into a series of deep folds, which run from top to bottom on each side. These folds, five in number, eventually reach through the mesoderm to the ectoderm, and fuse with it. Again, the combined ectoderm and endoderm breaks down and forms an opening. In this case, the openings correspond to the gill openings offish; in the higher groups of vertebrates they make only a transitory appearance during early embryonic life, before becoming transformed into something else; in some cases they never open com- pletely at any stage. Their fate in the various groups is too complex to be followed in detail here. (For instance, the most anterior forms part of the Eustachian tube and the tympanic cavity of the middle ear.) Between each of the gill slits (which are also known as pharyngeal or visceral clefts) is a gill 'arch'. In these there is a core of mesoderm between the ectoderm and endoderm, and in the centre of this core, an important blood vessel, one of the so-called aortic arches, will eventually run. Essentially the same structures are formed in the chick, but by somewhat different processes, since the anterior part of the gut, as we have seen (p. 252) appears not during gastrulation but in connection with the head fold. The formation of the mouth and pharynx, however, involves the same processes of the local fusion of ectoderm and endoderm, followed by their breaking down to give place to an opening. In the cliick, another derivative of the pharynx makes its appearance at a fairly early stage. A pocket of endoderm pushes out from the floor towards the posterior end of the swollen pharyngeal region, and rapidly elongates and extends backwards; it soon branches into two. This is the rudiment of the trachea leading to the two lungs. In the Amphibia it arises in the same region and in a similar way, but at a considerably later stage. 258 PRINCIPLES OF EMBRYOLOGY 4. The trunk: ectodermal organs As might be expected, it is in the main body of the embryo that the three fundamental layers, of ectoderm, mesoderm and endoderm, are most typically developed and most clearly defmed. The ectoderm produces only three main organs or organ-systems. The mid-dorsal part forms the central nervous system, v^hich in the trunk is in the shape of a tube with thick walls, a thin floor and roof, and a cavity wliich is high and narrow in transverse section. From this tube the ventral motor roots grow out in a series corresponding to the somites. The dorsal sensory roots, with the accompanying ganglia, although they are even- tually so closely associated with the main central nervous system, have a different origin in that they arise from the neural crest material. This, the second of the three ectodermal systems, also comprises the sympathetic nervous system and contributes to the mesodermal sheaths of the spinal cord; moreover the greater part of the pigmented cells of the body, whether they lie in the skin or in the linings of the gut and other internal organs, arise from the same source (Rawles 1948, 1953) (the most im- portant pigmented tissues with another origin are those of the iris and outer layers of the eyeball). Finally the third system formed from the ectoderm is the outer layer of the skin, the epidermis. The skin as a whole is a composite structure, a major part of its thickness being contributed by the mesodermal layer known as the dermis, which originates from the upper layers of the somites. The skins of different classes of vertebrates are by no means simple structures, but contain several sorts of sweat glands, oil glands and so on. Two of these structures are of particular importance, and are worth mentioning very briefly, namely feathers and hairs. Feathers are formed from a so-called feather germ or follicle. This is a slight hillock on the skin, from the tip of which a canal extends down through the thickness of the follicle. At the base of the canal lies a conical papilla, and it is from this that the feather actually grows. The papilla is a double structure, with an epidermal (ectodermal) cap fitting over a dermal (mesodermal) core. Many experiments have been performed on this structure, since it is one of the few organs in a bird which will continue to go through its develop- mental performance late into adult life; when a feather is plucked it will be regrown from its original follicle. Wang (1943) was able to peel the epidermal cap away from the core, and transplant the latter into follicles from which their own papillae had been removed. He showed that the dermal core can induce such a follicle to produce a new epidermal cap, and eventually a feather. Tliis is an example of a 'secondary' organiser ORGAN DEVELOPMENT IN VERTEBRATES 259 acting at a very late stage. The induction is not effective on non-follicular ectoderm; and it is very remarkable that when a feather is induced in this way, the details of its structure (for instance, whether it is typical of the breast or the back) is determined not by the dermal core which in- duced it but by the place of origin of the follicular epidermis which responded to the inducing stimulus. (For a longer discussion of feather formation see Waddington 1952a). Hairs also are formed from follicles which possess both ectodermal and mesodermal components. Much less is known about their structure and the inducing actions, if any, which go on within them; but since one special type of hair, namely wool, is one of the major raw materials of industry, a great deal is known about its development in other respects. Perhaps the aspect which is of most interest to general embryology is the study of the various shapes which may be taken by wool fibres, a subject which has been largely opened up through the pioneer investigations of Dry (1933-34). There are many different types of wool fleeces; each of them is characterised by a particular array of fibre-types, which occur with particular frequencies and can be distinguished by their length, thickness and the sequence of curves along them. It has long been known that the follicles occur in groups in the skin; within each group, the fol- hcles develop in series, first the central primary, then the lateral primaries, finally one or two waves of secondaries. Fraser (1952) has recently pre- sented evidence that each type of fibre is formed from some particular type of foUicle; and he has elaborated a theory which shows how one could account for the shapes of the fibres, which differ in the series of curvatures along their length, by the interaction between a varying growth rate and a regular periodic chance in the direction in which the fibre is pushed out. If all the fibres grew at a constant rate, they would all have a regular wavy form. But Fraser suggests that actually they grow at a rate which is dependent on the efficiency of their follicle in competing with other follicles for a limited quantity of available substrate; and this efficiency is supposed to change according to the time of origin of the folhcle. In the various breeds of sheep there are differences, not only in the relative numbers of the primary and secondary follicles, and in their density per unit area, but also in the curve which relates the efficiency to the time of origin of follicle (Fig. 12.4). In this way, the different shapes of the fmal structures can be accounted for in terms of physiological processes. The theory, although still in need of further testing, is a good example of the kind of mechanism one has to search for in the attempt to explain the facts of structure in terms of functional activities. The formation of hairs, which usually grow very rapidly in comparison 26o PRINCIPLES OF EMBRYOLOGY with the cells nearby, would seem to provide a good opportunity for studying the processes of protein synthesis in embryos. Some histo- chemical work on the growth of wool has been pubhshed by Hardy (1952), who finds that, as usual, RNA is present in high concentration in the cells in which growth is occurring most rapidly. The distribution of some other substances thought to be important for protein synthesis, such as ssss ssss Birth Figure 12.4 The rate of growth of the wool fibre from a follicle depends on (i) the amount of available substrate, which is proportional to the area of skin, (2) the efficiency of the follicle divided by the sum of the efficiencies of all the other follicles with which it is competing. In the figure on the left, the time of initiation of the primary [Pc and Pi), and secondary (S) follicles is shown below. The dotted line shows the increase in skin area. The group of dashed lines show various relationships between the efficiency of a follicle and its time of initiation — the upper curve being that characteristic of a coarse fleece, the lower one of a fine fleece. The stepped line shows the increase in the total efficiencies of all the follicles. The figure on the right shows the growth curves of a series of fibres calculated on this basis (medium fleece). (After Fraser 1952.) sulphydryl-containing proteins, is also interesting, but the whole range of data cannot yet be fully interpreted (Fig. 12.5). Investigations by Lees and Picken on the influence of genetic factors on the rate of synthesis and the kind of protein produced in Drosophila hairs are referred to on p. 337. 5. The trunk: endodermal structures It is, of course, the endoderm which forms the innermost structures, namely the gut and its annexes. In fact it produces only the central core of these, since the adult organs include an investment of mesoderm which clothes the original endodermal rudiments. We have seen that in the Amphibia the process of gastrulation itself ORGAN DEVELOPMENT IN VERTEBRATES 261 produces a tubular primitive gut reaching from the blastopore, which occupies the position of the anus, to the head. It was for long beheved that the cavity of this remained as the lumen of the adult gut, and that the varioiis organs associated with the gut developed as outpocketmgs from Its walls. The most strikmg of these pockets develops just posteriorly to the pharynx, and is usually considered to be the rudiment of the liver Accordmg to recent studies by Balmsky (1947), however, this convention- al mterpretation is in error. He claims to have shown, by vital staining Figure 12.5 Semi-diagrammatic longitudinal sections of hair follicles in the mouse. On mk RNr^r '" K " -Ipl^ydryl groups, on the right to show cytoplas- mic RNA. S.G., sebaceous gland; D.P., dermal papilla. (From Hardy 1952 ) experiments, that the so-called liver pocket continues to elongate bac wards until it eventually reaches right back to the blastopore region In fact It constitutes the whole of the defmitive gut, and outfoldings which wi give rise to the liver, the pancreas and other organs appear in its walls, not in those of the primitive gut, which eventuaUy disappears completely. These conclusions relate to Urodela. Nakamura and Tahara icture '"^ ^'''''^ ^^^ ''^''^^'°'' '' """""^ ^"^^ ^^^ conventional In the chick the rnain stretch of intestine (the midgut) is formed by the contmued backward extension of the pocket of the foregut. A similar 262 PRINCIPLES OF EMBRYOLOGY pocket, to be mentioned later, is formed under the tail, originating at a later stage than the foregut. It slowly extends forwards, and the two gradually come together near the middle of the body, leaving a narrow coimection through which the gut opens out on to the underlying yolk; this is the umbilical cord. It is not till near the end of incubation that the chick gut becomes completely closed, and this is achieved, not by closing off the umbilical cord, but by the small remaining piece of yolk being drawn in inside the body, when the ectoderm and mesoderm close the hole through which it has entered. 6. The trunk: mesodermal structures The mesoderm makes up the greater part of the bulk of the adult body, between the outer ectodermal layer of the skin (the epidermis) and the iimer endodermal layer of the gut (the intestinal lining). The first organs wliich become separated out from the rest of the meso- derm are those lying along the dorsal side. Immediately under the centre of the neural tube, a long rod is formed; this is the notochord, which acts as the first element in the skeleton, providing a longitudinal stiffening of the axis of the body. Its stiffness is not due in any large part to the pro- duction of hard or inflexible substance, but to the fact that the cells be- come swollen with fluid, so that the whole rod becomes turgid and stiff. The principle is one which Nature has made use of in other cases, where a temporary stiflhess is required for physiological functioning. In this case the turgor is more permanent since the notochord induces the neighbour- ing mesoderm to secrete around it a thin but inelastic sheath (Mookerjee 1953). On each side, the layer of mesoderm is thicker at its median edges, where it abuts on to the notochord. Very soon, this thicker portion becomes more or less separated from the more lateral parts by the forma- tion of a thinner longitudinal strip; this is known as the intermediate mesoderm, and from it the kidneys will develop. The thicker medial strips of mesoderm soon become cut up by a series of transverse grooves into separate blocks, known as the somites, which lie in pairs on each side of the notochord. The transverse grooving, and thus the appearance of the distinguishable somites, begins at the anterior end, and gradually progresses posteriorly, until there may be forty or more pairs of somites; the number differs in different species. One or more of the most anterior pairs may break up and disappear fairly shortly after their formation. The remainder of the somites persist and gradually give rise to the main segmental organs of the trunk, particularly the vertebrae and the associa- ted segmental muscles ; they also contribute to the dermal layer of the ORGAN DEVELOPMENT IN VERTEBRATES 263 skill. Somewhat unexpectedly at first sight, the boundaries between the vertebrae do not correspond to the grooves between the somites ; in each vertebra of the adult the posterior half has been produced by the anterior part of one somite while the anterior half is derived from the posterior part of the somite next forwards in the series. This arrangement ensures that the muscles arising within each somite are from the beginning joined to two contiguous vertebrae. In Amphioxus, and in the primitive vertebrates such as cartilaginous fishes, the somites are hollow (Fig. 12.7). The cavity within them (the myocoel) is part of the general body-cavity, and is continuous with the larger and better-developed space (the splanchnocoel) which forms within the lateral mesoderm. Both cavities together are known as the coelome. In higher vertebrates the myocoels are small and not always easy to detect. It is within the lateral parts of the mesoderm that the main body- cavity of the adult develops; the mesoderm lying above the space be- comes closely applied to the ectoderm, and forms the dermal layer of the skin, while that below lies against the endoderm and produces the muscular layer of the gut. The connection between the upper and lower layers of mesoderm persists in certain places, and provides the mesenteries by which the gut and its derivatives are attached to the main part of the body. 7. The tail and hind part of the body At the stage when, in the amphibian, the neural plate becomes clearly dehmited and the neural folds appear, the greater part of the plate is destined to form the brain and the nervous system of the anterior region of the body. The material for the whole posterior part of the trunk, and for the tail, is concentrated in a small region near the remains of the blasto- pore. In the chick, the material for the brain and anterior end arrives in place, and begins to differentiate, still more in advance of that destined to build the posterior end; and by the time only five or six pairs of somites have appeared, the primitive streak has already become quite short, although the greater part of the trunk is still to be produced. These facts have suggested to some authors, of whom the most authoritative in recent years was Holmdahl (1939), that the gastrulation process as we normally conceive it is responsible only for the formation of the anterior part of the animal and that the posterior part is produced by some radi- cally different process which goes on within the small remnant of blasto- pore or primitive streak. Tliis region, from which the posterior part forms, is knov^Ti as the tail-bud. The authors who argue that the processes going on within it are quite different from those of gastrulation nevertheless 264 PRINCIPLES OF EMBRYOLOGY do not agree on the position of the dividing hne between the anterior part and the posterior part for which it is responsible. The existence of this theory has led to an intense study of the develop- ment of the region in the neighbourhood of the blastopore of the neural- plate stage amphibian. Bijtel (193 1) first showed that invagination is still proceeding at the blastopore even after the appearance of the neural folds; and further that some of the material between the posterior ends of the folds (i.e. material of the neural plate itself) will actually form mesoderm (Fig. 9.10, p. 166). More recent authors, particularly Pasteels (1939), Nakamura (1942) and Chuang (1947) fully confirm this, and demonstrate conclusive- ly that the processes going on in the late blastopore are essentially invagin- ation processes broadly similar to those of gastrulation proper. The main differences are two. The first is relatively trivial. Throughout the earlier phases of gastrulation, the dorsal midline above the blastopore is occupied by presumptive notochord. hi the neurula stage, however, the last piece of presumptive notochord invaginates before all the somite mesoderm has moved into the interior; and in its final stages, therefore, the dorsal lip of the blastopore consists of presumptive somite material. The second difference is perhaps more important. Combined with the normal in- rolling movement of gastrulation, there is in the late blastopore a stretch- ing by wliich the tail is thrust out as an elongated structure. This produces a rather complicated system of movements, but it does not alter the essential fact that each region of tissue moves in a precisely defined way and reaches a definite fmal position. There is no reason to believe, as we are urged to do by Holmdahl, that the tail-bud is a mass of in- different tissue from the general undifferentiated bulk of which the posterior neural tube, somites and chorda appear. In the chick, the precise movements occurring in the late primitive streak are not so fully known, but there seems no reason to doubt that here again the posterior part of the body is produced by gastrulation pro- cesses essentially similar to those which give rise to the anterior end (cf. Pasteels 1939, Waddington 1952^). Associated with the tail-bud is the hind end of the gut. This opens to the exterior through the anus, or cloaca, which is formed near the site of the blastopore, but not directly out of it. In forms such as Amphibia, which during gastrulation possess an open blastopore leading in to the cavity of the gut, this opening is closed by the fusion of its lips as invagina- tion terminates. From the hind end of the gut, two pockets are then pushed out. One extends from the ventral side of the gut, and this reaches the ectoderm (which may fold inwards to meet it) ; the endoderm and ecto- derm first fuse, and then break down to give an opening which becomes ORGAN DEVELOPMENT IN VERTEBRATES 265 the definitive cloaca and anus. The other endodermal pocket starts from the dorsal side of the gut and extends backwards into the tail, where it is known as the tailgut. The extent to which it is developed varies greatly in different species even of the same group of animals; thus in the frog it is inconspicuous, while in some toads it forms a rather long tube, which extends right round the posterior tip of the notochord, its lumen becom- ing confluent with the cavity of the neural tube (the junction being known as the neurenteric canal) (Fig. 12.6). Mesoderm Figure 12.6 On the left, a diagrammatic longitudinal section through a newt neurula. Closely lined neural plate, showing the future regions into which it will develop; darkly dotted, notochord; lightly dotted, endoderm; cross-hatch- ed, ventral mesoderm. On the right, section tlixough the tail region at a later stage. 8. The kidneys There are three main types of 'kidney' developed in vertebrate embryos. First, the pronephros forms in a more or less anterior position; later the mesonephros appears further posteriorly; and fmally the metanephros differentiates still further back. All these organs are paired, one appearing on each side of the body in the so-called 'intermediate' mesoderm, i.e. that lying between the somites and the lateral plate (Fig. 12.7). The basic unit, from which the kidneys are constructed can be thought of as a tube, one end of which opens into the coelome, while the other is con- nected with a duct -which leads the secretion away towards the posterior; and somewhere between these two ends, the tube becomes closely ap- posed to a blood vessel. In functional kidneys, this simple unit becomes highly modified and comphcated; we shall not discuss these details which belong rather to the province of comparative morphology than that of general embryology. But it is necessary to say something about the duct. 266 PRINCIPLES OF EMBRYOLOGY This is a tube which leads, in the early stages, from the pronephros to the posterior where it opens into the cloaca. It is at that time known as the pronephric duct. Later the meson ephric tubules become connected with it; and in the higher vertebrates the pronephros degenerates and disappears; the duct is then entitled to be called the mesonephric duct. (It is also re- ferred to as the Wolffian duct.) Finally, a diverticulum is pushed out from it, starting from the region near the cloaca; this makes contact with Neural crest Figuiie"i2.7 Diagrammatic section through the anterior trunk of a vertebrate, showing the relations of the mesonephric tubules. the metanephros, and is known as the metanephric duct (Fig. 12.8). Closely associated with it is another duct, the Mullerian duct; this leads from the exterior to the gonads, which originate near the kidneys, though they may later shift into another region of the abdomen. The comparative study of kidney formation in the different classes o. vertebrates, besides being of great interest from the anatomical point of view, illustrates one or two points of importance for general embryology. In the first place, we may note that the pronephros, which is an actively functional excretory organ in amphibian tadpoles, is formed opposite a well-defined group of somites; numbers 2, 3 and 4 in Anura, and num- bers 3 and 4 in urodeles (cf Cambar 1949)- hi the chick, in which the pronephric tubules are rudimentary, and never function as excretory organs, they are developed transitorily opposite somites 5 to 16. This is a good example of an organ being moved along the length of the body ORGAN DEVELOPMENT IN VERTEBRATES 267 during evolution, a phenomenon which is of fairly widespread occur- rence. The epigenetics of kidney development has been rather extensively studied; recent reviews are those of Fraser (1950) for the vertebrates in general, Cambar (1948) for Amphibia and Waddington (1952^) for birds. In the Amphibia, the capacity to develop into pronephros appears at a certain level of a gradient which runs from a high point in the chorda to a low in the lateral plate. This was demonstrated by Yamada (1940), who showed that if lateral mesoderm is taken from a position in the neurula some distance away from the embryonic axis and cultivated in isolation, it will develop only into tissues normally formed from such lateral regions, such as blood, while if chorda is added to the isolate, pronephric tubules appear (see Fig. 10.10, p. 191). It is to be presumed that the first step in the development of the more posterior intermediate mesoderm (which will form mesonephros) is taken in the same way; and it seems not unlikely that a similar process occurs in the bird embryo, though this has not been defmitely proved (cf. Waddington 1952^). There is no evidence that the amphibian pronephros requires any further stimulus, after its position on the medio-lateral gradient is fixed, before being able to complete its development. For the more posterior kidneys (mesonephros and metanephros), however, something further is necessary, namely an inductive influence which is normally exerted by the pronephric duct. As we have seen, this duct grows backwards from the region of the pronephros. If its backward extension is prevented (e.g. by a transverse cut which fails to heal completely) no sign of the duct appears in the posterior region, and only minor traces of the mesonephros develop. The dependence of the amphibian mesonephros on an inductive stimulus from the pronephric duct was first suggested by Miura (1930) and later work by many authors has fully confirmed it (O'Connor 1939, Cambar 1948). In birds, Boyden (1927), Griinwald (1937) and Waddington (1938) have found a similar situation (Fig. 12.8). In birds the pronephros never functions as an excretory organ, and Waddington suggested that it had been retained in the ontogeny of the animal simply because it is an essential step in the formation of the prone- phric duct which is itself necessary as the inducer of the mesonephros. Cambar (1948) has criticised tliis suggestion on the grounds that in the Amphibia the pronephric duct arises from a mass of tissue wliich is dis- tinct from, although it lies immediately in contact v^th, that which gives rise to the pronephros itself; moreover he states that the growth of the duct is independent of the continued presence of the pronephros, whence 268 PRINCIPLES OF EMBRYOLOGY he concludes that that organ is quite unnecessary for the production of the mesonephros. However, it is not at all clear that even in the Amphibia the original production of the rudiment of the duct is quite independent of the presence of the pronephros, and in birds the two components are so intimately associated that it is difficult to imagine the duct being pro- duced without at least some transitory appearance of the tubules of the yM Wd? =«0 G 1 50 ^ •^ «<0 1 «0-M ^ V'i ^ •*•■■; :«0 /•.*; f>C '•{}. 1 W.D M. Figure 12.8 Figure 1 shows, on the left a diagram of the normal urogenital system, com- prising the adrenal {A), gonad (G), mesonephros (M), MuUerian duct {MD), WohFian (or mesonephric) duct {WD), ureter {U) and definitive kidney (metanephros) (iC). The parts which develop independently of others are shown in solid black, those resulting from inductive actions in outline. On the right is the result of preventing the mesonephric duct from growing posteriorly beyond the arrow; the posterior mesonephros and other organs fail to appear. (From Gruenwald 1952.) Figure 2 is a section through a chick embryo in which the posterior growth of the pronephric duct was prevented on the right side. There is not only no sign of the duct [WD] but the meso- nephros {M) has also failed. (From Waddington 1952^.) nephros. There seems, therefore, no good reason to doubt that we have, in the appearance of the pronephros in birds, an example of the retention during evolution of an organ for the sake of its function as a component of the epigenetic system, rather than for any contribution it makes to the physiological functioning of the embryo as a metabohsing organism. Although one may probably conclude that the pronephric duct is an important factor in the epigenetic system, it is by no means the only actor on the stage. The competence of the intermediate mesoderm plays ORGAN DEVELOPMENT IN VERTEBRATES 269 an important part. Even in the absence of the duct, accumulations of what one may consider as 'pre-mesonephric' cells may put in a transitory appearance. Further, the inductive influence of the duct is only effective if it operates on intermediate mesoderm (v^hich has probably been brought to a state of competence by its position in the medio-lateral gradient) ; and this mesoderm can, in the chick at least (Griinwald 1942, 1943), react suc- cessfully to abnormal inductors, such as transplanted pieces of neural tissue. Griinv^ald also showed that an inductive reaction is concerned in the production of the metanephros of the chick. Here the inductor is the diverticulum which pushes out from the region where the proncphric duct joins the cloaca. He discovered the remarkable fact that if a piece of the main (mesonephric) region of the duct is substituted for this diverti- culum, and allowed to act on the presumptive metanephric tissue, it succeeds in inducing kidney, but mesonephros rather than metanephros. This is one of the comparatively few cases in which the character of an induced organ is determined by the nature of the inducer rather than by the competence of the reactant. Grobstein (195 3 rt, h) is analysing the inductive reaction between the duct diverticulum (uretic bud) and the metanephric mesoderm in the mouse, which is probably rather similar. He fmds that the mesoderm can be induced to form tubules by a variety of different types of tissue, all of which are epithelial in character and to that extent at least similar to the uretic bud which is the normal inducer. An important result is that the inducing agent given off by embryonic spinal cord, for example, can pass through a 20[jl thickness of an artificial porous membrane; this is one of the most direct proofs that induction may be carried out by diffusible chemical substances. Grobstein has used the same methods for investisat- ing other examples of the development of glands which contain both epithehal and mesenchymal components, particularly the submandibular (salivary) gland. By careful exposure to trypsin solutions, he can separate the epithelial and mesenchymal tissues; the epithelium can then be cultured in combination with mesenchyme from its own type of gland or with that from some other organ. He fmds that the epithelium differ- entiates typically only when combined with its own type of mesenchyme; other mesenchymal tissues may partially inhibit the spreading tendency which is usually seen in isolated epithelium, but do not induce the forma- tion of normal tubules. If the epithehum and mesenchyme are separated by a fme-grain fdter membrane, the inhibitory action of foreign mesen- chyme passes through, and the specific effect of like mesenchyme also does so to some extent, though not completely; it induces the formation of tubules but not quite typical ones (Fig. 12.9). 270 PRINCIPLES OF EMBRYOLOGY The reproductive organs of vertebrates develop in rather close associa- tion with the kidneys and their ducts. We shall not deal with them here; recent summaries of the literature may be found in Nelsen (1953) and Nieuwkoop (1946). The germ-cells themselves originate at considerable distances from the glands in which they eventually lie, and reach them after performing a peculiar migration, as isolated mobile cells, through the intervening tissues of the embryo. The place of their origin seems to be surprisingly different in different groups ; probably the lateral Figure 12.9 A shows the epitheUum of the submandibular gland of the mouse growing in tissue culture combined with mesoderm from the same source: the epi- thelium is forming typical tubules. In B the epithelium is lying on top of a porous membrane, below which is mesoderm from another source (maxil- lary region) : no morphogenesis. C is a similar culture but the mesoderm below the membrane is from the submandibular gland: tubule formation has been induced, although the morphogenesis is somewhat abnormal. (After Grobstein 1953.) mesoderm in urodeles, the posterior dorsal endoderm in Anura, endoderm lying anterior to the head in birds, yolk-sac endoderm and mesoderm from the posterior end of the primitive streak in mice. An introduction to the literature can be found in Nieuwkoop (1949), Willier (1950) and Chi- quoine (1954). 9. The limbs The limbs first appear as slight external swellings which fairly rapidly elongate. In their early stages they consist of condensations of mesoderm ORGAN DEVELOPMENT IN VERTEBRATES 27I covered by epidermis which does not differ from that of the rest of the body. The mesoderm cells at first show little sign of particular differen- tiation, but form a rather loosely aggregated mass of mesenchyme. As the limb elongates, the cells towards the centre of it become more tightly packed, forming a number of condensations within the mesenchyme (Fig. 20.5, p. 429V These group themselves into the pattern of the skeletal elements of the normal limb, and gradually differentiate, first into cartil- age and then into bone. Meanwhile the remaining mesenchyme develops into the muscles. The development of the limbs has been very extensively studied, and provides examples of a number of points which cannot be so well illus- trated in any other field. At about the time of the First World War, Ross Harrison (191 8) began a long study on the polarity and asymmetry of the limbs of urodeles. The subject was also pursued by a number of his students, such as Detwiler and Swett. The most recent summary of the extensive literature of this group of workers is that of Swett (1937) and the main contribution since then is an extensive and important work by Takaya (i94i)- It is clear that a fully developed limb must be either a right or a left limb and that these two have essentially different asymmetry, being, in fact, mirror images of one another. The genesis of tliis asymmetry can be studied by excising the presumptive limb region from an embryo and grafting it back in such a way as to change the relation between the polarity of the graft and that of the host body. Consider, for instance, the forehmb of a newt. In the tail-bud stage the region from which this limb will develop is represented by a circular area on the side of the body just below somites 3 and 4. Suppose that the limb area on the left side of a newt embryo was cut out, then pushed up to the dorsal midline and down the other side, and eventually grafted in place of the right limb area, which had been previously removed. Then it is clear that its anterior end would still point towards the anterior end of the whole body, and its exterior side would still lie towards the exterior, but its dorsal side would now be below and its original ventral side uppermost. Such an orientation is described by saying that we have reversed the dorso-ventral axis of the graft but left its antero-posterior and medio-lateral axes unchanged. We could, of course, reverse both the antero-posterior and dorso-ventral axes leaving the medio-lateral one unchanged, by making a circular cut around the limb-forming area on one side and then rotating the area through 180 degrees about an axis perpendicular to its surface before allowing it to heal in again. By a variety of such methods one can, in fact, reverse at will any particular axis or combination of axes. After such 272 PRINCIPLES OF EMBRYOLOGY Operations one allows the limb to develop and examines whether a disc whose antero-posterior axis was reversed actually develops back to front, in which case the axis may be said to have been already determined, or whether influences from the host's body succeed in causing a reversal of polarity within the limb-disc (Fig. 12.10). It was soon discovered that the various axes are determined at different times. The antero-posterior one always becomes fixed first. According to Detwiler it is already determined by the middle gastrula stage, but Takaya gives reasons for doubting this, and suggests that the determina- tion does not actually occur until the neural plate stage. Even so, this is much earlier than the determination of the dorso-ventral axis, which does Figure 12.10 On the left, an axolotl embryo into which a hmb-bud has been grafted with its dorso-ventral and anterior-posterior axes reversed. The transplant [Tr) has developed into a limb with reversed anterior-posterior axis but normal dorso-ventral axis. (After Harrison.) On the right, a newt embryo showing the direction of the anterior-posterior field in different regions of the flank. The circles mark the normal positions of the limb-buds. (From Takaya 1941.) not occur till the fairly late tail-bud stage, by which time the limb-buds have already become slightly elevated from the side of the body. It is shortly after this that the medio-lateral axis of the mesodermal com- ponent of the limb-bud becomes also determined. The determination is certainly brought about by the tissues in the im- mediate neighbourhood of the disc. This can be shown by rotating a fairly large area in the limb region and then grafting into this region a limb- disc, with one or more of its axes reversed. In such cases it is the orienta- tion of the immediately surrounding area rather than that of the whole host embryo which is decisive over the future development of the limb. The nature of the influence which fixes the polarity of the limb-disc is still imperfectly understood. It clearly falls into the general category of what have been referred to as 'field characters', but that terminology does not tell us much about what it actually is. Harrison (1936) thought that the polarity might depend on some fme-grain structure of the tissue of ORGAN DEVELOPMENT IN VERTEBRATES 273 almost molecular dimensions and perhaps comparable to the oriented arrangements found in liquid crystals, but x-ray microscopy was not able to detect any such structure (Harrison, Anthony and Rudall 1940). Takaya, on the other hand, suggests that the fundamental factor is capacity for growth, which is highest in the antero-dorsal part of the early limb-disc. In his view it is the fixing of gradients in growth capacity which deter- mines the polarity which the limb will exhibit. If an early limb-disc is excised and grafted into a new position in such a way that its polarity is opposed to that of the immediately surrounding area, a frequent result is the development of a pair of duplicated limbs. These are nearly always mirror images of one another, one having the symmetry of a right limb and the other that of a left. The frequency with which this mirror-imaging occurs suggests that two limbs developing close to one another influence each other's polarity. This is confirmed by the results of grafting two limb-buds into each other's neighbourhood. It is found that even if they are grafted so that their polarities are con- cordant, nevertheless the two limbs which form often turn out to be mirror images, one of the two having had its original polarity reversed by its neighbour (Fig. 12.11). It is a remarkable fact that the polarity of a normal embryo does not run in a constant direction throughout the whole flank of the animal. Opposite the anterior somites, from somite i to about somite 7, the polarity is such that it tends to cause the pre-axial side of the limb to develop on the side nearest the head of the embryo. The same is true of posterior regions where the hindlimb forms, from about somite 14 back- wards, but in the midflank region, from the level of somite 7 to that of somite 14, the polarity of the flank is in the opposite direction (Takaya 1941) (see Fig. 12.10). This reversal of polarity in the flank is clearly expressed in the experi- ments which have been made on the induction of limbs. Tliis was first successfully accomplished by Balinsky (1925). He found that if an ear vesicle is transplanted into the flank of a young tail-bud embryo it induces the formation of a supernumerary limb. Later work (Balinsky 1933) showed that the same effect could be produced even more regularly by the implantation of a nasal placode. The organs used in these grafts can not, of course, be the normal inducer of the limb. It is not at all clear what organ or tissue fulfils tliis function in normal development. Attempts to induce limbs by the implantation into the flank of the pronephros, which lies near the site of the normal forehmb, have so far been unsuccessful. The induction of limbs by such 'foreign' tissues as the auditory and nasal vesicles, raises in an acute form the problem of the specificity of the 274 PRINCIPLES OF EMBRYOLOGY inducing stimulus. Do the inducers give off some specific substance which IS essential for limb formation, or do they only activate potentialities already fully present in the flank? Needham (1942) argues that the former alternative is the more likely on general grounds and is the better guide to future experimental w^ork. Balinsky (1937), on the other hand, suggests that the inductors owe their power to a rather ill-defmed quality which he speaks of as a 'high physiological activity', and he beheves that it is this which sets in motion the inherent capacity of the flank mesenchyme to develop into a limb. The question needs much further study; no serious attempt has yet been made to investigate the phenomenon at a biochemi- cal level. It seems likely that it will turn out to be very similar to the situa- tion with which we are confronted in the induction of the neural plate : Mirror imaging when limb-buds develop near one another. In a the ap axes are concordant; one of the limbs frequently has its polarity reversed. In b the ap axes point away from each other, and both limbs retain their polarity. In c the ap axes point towards one another, and a supernumerary limb, of ill-defmed polarity, often appears. (From Takaya 1941.) that is to say, that the induction can be performed by relatively unspecific implants, but that these may act in a secondary way, their first effect being the release of specific substances within the reacting tissues. One single case has been described [Bahnsky 1927] of the induction of a hmb follow- ing the implantation of a fragment of celloidin into the flank. This may probably be compared with the induction of neural plate by treatments which produce 'sub-lethal cytolysis' (see p. 196). Whatever the position as regards the specificity of the inducing stimu- lus, there is no doubt that the competence of the reacting material plays a large part in the production of the limb. One evidence of this is the asym- metry of the induced limb in the various regions of the flank. Back to the level of somite 7 the pre-axial side develops anteriorly, but between somites 7 and 14 the asymmetry is reversed (Takaya 1941). Again Balinsky (1933) showed that the frequency with which limbs are induced falls oft fairly steadily from the region of the forehmb- to that of the hindlimb-bud ORGAN DEVELOPMENT IN VERTEBRATES 275 (Fig. 12.12). This gradient is probably not a straightforward reflection of the intrinsic capacities of the flank mesenchyme, but is partially a result so ¥0 20 10 Figure 12.12 Limb induction in the newt. The graph above shows the frequency with which hmbs (thick line) or pelvic girdles (thin line) were induced by the implantation of nasal placodes in the corresponding region of the flank indicated on the drawing below. In the drawing the position of the normal limbs is shaded, that of the pronephros dotted. (From Balinsky 1933.) of the general influence of the body as a w^hole. Thus Takaya has shov^n that if material from the lowest point in the gradient (just in front of the hindlimb-bud) is transplanted to a more anterior position, and then sub- mitted to the influence of an implanted inductor, the frequency of limb 276 PRINCIPLES OF EMBRYOLOGY induction is much higher than it would have been if the material had been left in its original location. Another manifestation of the field of competence can be seen in the frequency with which the induction produces forelimbs or hindlimbs. Forelimb-hke structures can be induced in the anterior region back to the posterior margin of somite 8, while hindlimbs can be induced in the posterior region, wliich extends forward to the anterior margin of seg- ment 8. Thus there is a small region of overlap between the forelimb- and hindhmb-producing regions. Shoulder girdles are usually not induced, but the pelvis can be evoked very regularly in the posterior end of the field near somites 13 and 14. These three lines of evidence — the asymmetry of the induced limbs, the frequency of the induction and the character of the limbs induced — clearly demonstrate the existence of a field of competence in the flank, but do not show how far this is a property of the localised areas of tissue, or how far it is dependent on factors which act in a general way over the whole region. In the period immediately after the limb-bud is formed, the various individual parts of it are still incompletely determined. A single bud may give rise to a duphcated pair of limbs, or, in other cases, two anterior half-buds may fuse to give a single limb. We shall not carry any further the discussion of the gradually increasing determination of the ampliibian limb-bud (see Review of Mangold 1929), but instead turn to a considera- tion of the limb-buds in the chick, which illustrate certain other points of general significance. In the chick, technical difficulties have made it impossible to investigate the symmetry relations of the early limb-discs as has been done in the Amphibia, nor is anything known about limb induction in birds. It is in connection with the later stages of development of the limbs that investigations on bird embryo have been most informative. The first point we shall notice is one which emerges from the investigations of Saunders (1948). He showed that the bulk of the material which forms the limb-bud at the earliest stage at which it can be recognised will eventually form the proximal parts of the limb. More distal parts are added later as the limb-bud elongates. During this process a specially important part is played by a thickened cap of ectoderm which forms the actual apex of the elongat- ing bud. If this apical cap of ectoderm is removed, the laying down of fur- ther distal regions of the limb ceases, and the limb remains as a stump from which the distal parts are missing (Fig. 12.13). This does not occur in the Amphibia, where the proximal parts can form a complete limb ; but in that group, of course, proximal stumps can regenerate their missing distal parts till quite a late stage, whUe such regeneration does not occur in the chick. ORGAN DEVELOPMENT IN VERTEBRATES 277 Young limb-buds of the chick can be excised from their normal site and planted into the coelom just lateral to the somites, where they will continue their development in a remarkably normal manner. In such situations the hmb-buds are often very incompletely innervated. In the nerveless limbs the muscles atrophy nearly completely, and the growth rate is somewhat less than normal, but although no movement at the v\ Figure 12.13 On the left, two stages (in section) of the Hmb-bud of the chick, showing the presumptive areas. C, coracoid; S, scapula; oblique shading, upper arm: dashes, forearm; horizontal shading, wrist and hand; A, apical ectoderm. On the right, the results of excising anterior [A above) or posterior (B below) regions of the apical ectoderm; the parts developing are shaded. (After Saunders 1948.) joints occurs, the structure of the skeletal elements is exceedingly normal, even to the formation of the joint surfaces (Hamburger 1939) (Fig- 12. 14). It is well known that if, owing to injury, a functional limb has to be moved in an abnormal manner, the joint surfaces may become modified in such a way as to facilitate this. It is clear, then, that function can play a part in moulding the structure of the skeleton, but the development of these isolated and functionless limbs shows that this structure can also develop with a considerable degree of perfection, solely under the influence o{ factors inherent within the developing tissues themselves. The interaction 278 PRINCIPLES OF EMBRYOLOGY of intrinsic and extrinsic factors in the architecture of the skeleton has been discussed particularly by Murray (1936). The development of Hmbs in abnormal sites in the body has provided the opportunity for a large number of investigations on the way in which the peripheral nerves make contact with the various parts of the limb, and also on the influence of an excess or deficiency of peripheral organs on the central nervous system There is no space here to do more than mention this subject as one which Figure 12.14 The development of a limb-bud of the chick, transplanted to the coelome and badly innervated {h), compared with that of a normal limb {a}, (brom Hamburger and Waugh I940-) provides evidence of epigenetic interactions between parts of the body continuing into the later stages of development. Recent reviews of the field will be found in such works as Piatt (1948), Weiss (1941, I950^) and Detwiler (1936). SUGGESTED READING Classical papers are Harrison 1918, 1936, 1945; and Spemann's work on the eye. summarised 1938, pp. 40-97- For a general survey of experimental work on Amphibia, Needham 1942, pp. 290-309; on the chick, Waddington 1952^, PP- 140-200, ^d Annals, Net. York Academy of Science 1952, Volume 55, Article 2. Two very interestmg Imes of work, hardly touched on m the main text, will be found in Rawles 1948 and Landauer 1954- CHAPTER XIII GROWTH I. Overall growth In everyday usage the word 'growth' is used to mean any type of in- crease in size. This is obviously one of the important phenomena in em- bryonic development and requires discussion. There have been two ways of approaching the problem; one is content to accept the everyday meaning of the word and to study the increases which take place in whole embryos or in their parts; the other has attempted to start from some more precisely defmed process of growth and to set up general norms from which the facts as they appear in the development of particular animals can be deduced as special consequences. This second attempt has not as yet proved very successful but it will be easier to exhibit the complexity of the whole situation if we start by discussing it (General Reviews : Needham 193 1, Medawar 1945). If we wish to consider a precisely defmed process of growth we shall have to fmd some way of limiting the concept so that it is confmed to the increase in size of something which retains a certain similarity to itself. Size may increase merely by the imbibition of water or by the laying down of relatively inert material such as shell, bone, cartilage, etc., and such processes obviously differ in kind from the increase in amount of the living material itself. Various definitions have been offered with the purpose of excluding them from the concept of growth as that is required for a precise theory. Gray (193 1) speaks of growth as 'essentially concerned with the formation of new living material'. Medawar (1941) states that 'what results from biological growth is itself typically capable of grow- ing'. Weiss (1949) gives a more formal dcfmition; growth is 'the in- crease in that part of the molecular population of an organic system which is synthesised within that system', and he further amplifies this, pointing out that it means 'the multiplication of that part of the molecular popula- tion capable of further continued reproduction'. This puts its fmger on the important point ; if we are trying to formulate a precise concept of growth we must confme it to the increase in the amount of the system which is capable of growing. The main general problem which then requires study is this — at what rate does this increase take place and how does the rate change as time passes ? The simplest possible situation would be one in which the rate of multi- plication per unit mass remained constant. We could formulate this T 279 PRINCIPLES OF EMBRYOLOGY dw 280 mathematicaUy by the equation ^ ^ = ^ constant (where w is the weight or mass of the system). This, of course, leads to the absolute size increasing ever faster and faster, at an exponential rate. The equation is solved to give w = 11^ oe^^ or log n^ = log Wo + ^^ when li^o is the initial size and fe is a constant. It is impossible to fmd a naturally occurring biological system which behaves so simply, and it is difficult to make one experimentally, though it can be done. If a smaU population of yeast cells, or bacteria, is moculated into a large mass of nutrient medium, allowed to grow for a time, and a new inoculum transferred to fresh medium after a fairly short period, the growth rate per unit mass may be kept constant indefmitely. The essential points are that neither lack of nutrient nor the presence of harmful excreta are allowed to inhibit the system. If frequent transfers are not made, one or other or both these are certain to occur, and the rate of growth will slacken tih the growmg mass becomes stationary and eventuaUy begms to dechne when the death-rate of cells overtakes the rate of mcrease. Samples taken from such declining cultures usually take a httle ttme to get going when transferred to fresh medium, so that m the typical growth curve of a colony of cells (yeast, bacteria, tissue-cultures and the like) the logarithm of size when plotted against time, is not a straight Ime, but has the form shown in Fig. 13.1. Figure 13.1 Typical growth curve of a population of isolated cells (e.g. yeast cells, bacteria, etc ) If the population starts with a group of cells taken from a non-growing colony, there is first a 'lag phase' (i) then a phase of exponential growth (2) in which the growth rate per unit mass is constant, then a phase of retarda- tion (3) when the medium is becoming exhausted and fmally a regression phase (4) when the medium can no longer support the population. GROWTH 281 The growth curve of a developing animal has a somev^hat similar form; although there is no definite lag before growth commences, the curve relating mass to time is sigmoid or roughly S-shaped. It was Minot, at about the beginning of the century and following him Brody, who particularly pointed out that it would be more profitable to consider the so-called 'specific growth rate' (i.e. growth rate per unit mass -, w dt which is the same as — -^— ) rather than the simple growth rate {dwidt) (Fig. 13.2). As we have just seen, it is only in exceptional circumstances that this can be expected to be constant. Many formulae have been ad- vanced, on a variety of grounds, in an attempt to produce a theoretical scheme which fits the facts better. Figure 13.2 Various 'growth functions'. The three curves in the left side are concerned with the absolute growth. In a weight is plotted against time to give the curve of growth (a Gompertz equation is assumed) ; in h growth rate is plotted against time, and in c the change of rate (i.e. acceleration) of growth is shown against time. The curves on the right give similar graphs of the functions of the specific growth rate ; that is, in e we plot 'growth rate per , 1 dW d\os,W . , r . , dW unit mass or— -j- = -j^ — mstead of simple growth rate --.-, and make corresponding changes in the other curves d and/. Notice that the specific growth rate falls steadily from the beginning of life (curve e), and does so at an ever-increasing rate (curve/). (From Medawar 1945.) 282 PRINCIPLES OF EMBRYOLOGY It is worth while just to glance at the main varieties of these. (i) The 'monomolecular' formula is derived from the idea that the growing substance is formed from a store of some substance a which is gradually used up. This gives the relation ^ k(a — tv), at whence i^ = 4^ - ^^"'')' when h is a constant depending on the initial amount of a. When time is plotted horizontally, this gives a curve which is convex upwards and which approaches the upper hmit a. Rather few growing systems behave in this way. r ^■rr ^ (2) The 'logistic' formula can be deduced from a number of difierent assumptions. For instance, if the substance lu is formed from a precursor a as before, but if the rate of formation is also increased m proportion to the amount o(w already present (i.e. if the reaction is 'autocatalytic ), we shall have dw J f \ dt Or, if we suppose that the rate of formation per unit mass decreases in proportion as w is formed, we shall have 1 dw , s w at where p and q are constant, which amounts to the same thing. The differential equation can be solved to give an expression of the form a ^ = r— ^ I + he- This when plotted gives a sigmoid curve, which eventuaUy approaches the limit a. (3) Another assumption is that^ -^ decreases as time goes on, but in a different way, being proportional to log w. This is the 'Gompertz equation : 1 dw ^ __ =p\ogw-q. tv dt This gives a solution of the form which is again a sigmoid curve which approaches the upper limit a. GROWTH 283 (4) Again, we can take it that — decreases simply in inverse propor- w at tion to the lapse of time 1 dw k w dt t' whence w = bt^. This is the 'parabolic' or 'double-log' curve; it should give a straight line when log weight is plotted against log time, whereas the 'exponential' or 'single log' formula lu = be^\ which holds when is a constant, gives w dt a straight line when log iv is plotted against time. The double-log formula fits better with the weights of such growing systems as embryos, at least in the early stages, but like the single-log expression, it has no upper limit as time increases and can therefore obviously only hold for part of the life-history of most animals, which reach a final adult size. (It is possible that certain animals, including fish, continue growing indefinitely.) All the formulae have been applied by various authors to the actual data derived by weigliing embryos during their growth. Such observed growth curves are generally roughly sigmoid in shape, but they may give evidence of a number of cycles of growth, so that applying any one type of formula one may have to invoke a set of different constants for each cycle. Even so, it has never been possible to show that any one of the above formulae fits the facts so exactly that it must represent the actual situation and the others can be excluded. There are many snags in fitting theoretical curves to the actual observations. Li the first place, many growth curves have been derived by weighing a sample population at each of a series of ages, taking the average of each age group, and joining the points together to give the overall curve. It is difficult to do anything else if we are inter- ested, for instance, in the foetal growth of a mammal. But there will, of course, be a certain variation in the stage of development reached by individuals of the same temporal age, and if there were any sudden spurts or slowings-down of growth these might be obscured by taking averages. For instance, there is usually a sudden spurt in the growth of a boy at the time of puberty. In some boys this occurs rather earlier, in some rather later. If one derives a growth curve by weighing groups of boys at various ages, the spurt becomes distributed among a number of different groups and its existence concealed. Even when a growth curve can be obtained by weighing a single indi- vidual at various times during its life, the curve is bound to suffer from a 284 PRINCIPLES OF EMBRYOLOGY certain lack of precision. There may have been unavoidable alterations in the environmental conditions, amounts of food, temperature, disease, etc., and each weighing will only be accurate within certain observational limits. Thus what we shall have as a basis for setting up the growth curve is not a set of absolutely precise points but rather a zone of greater or less width within which the curve must lie. It will always be possible to fit quite a number of different theoretical curves into such a zone, particularly if we allow ourselves to consider the possibility of a set of growth phases for each of which a new set of constants may be calculated. Thus it is extremely improbable, and in fact does not happen in practice, that a set of empirical observations can suffice to discriminate between the various theoretical possibilities. Moreover, it is quite obvious that when dealing with the growth of an entity such as an embryo, we are not confronted with a 'growth' which corresponds to any precise definition. The embryo contains a highly heterogeneous collection of tissues, some of which are growing, probably at difterent rates, while other parts of the embryo, such as the blood plasma, are not growing in any normal sense at all. Weiss (1949^) has given a very clear and vivid picture of the type of complexity which is involved, even in the growth of a single organ, such as the eye. He writes : 'The original eye vesicle consists of a certain initial allotment of cells from the embryonic brain wall. At first, all of these cells divide. The growth function at this stage is therefore a volume function. In the cup stage the retina becomes multilayered, with a sharp division into a germinal and a sterile zone. Only the cell layer in contact with the outer surface, corresponding to the ventricular (ependymal) layer of the brain, continues to proliferate, while the cells released into deeper layers differentiate the various retinal strata without further multiplication. The source of growth thus has become reduced to a two-dimensional one, causing a marked decline in the relative growth rate taken over the whole organ (e.g. from measurements of diameter). Later, the cells of the germ- inal layer themselves cease to proliferate and transform into sensory cells, a process which starts from the centre (macula) and spreads rapidly toward the periphery (cihary zone) of the retina. Eventually, only the cells at the rim retain residual capacity to multiply. Further growth is then essentially by apposition from this rim; that is, the growth source has shrunk from planar to linear extension. Meanwhile some of the neuroblasts, though no longer multiplying, grow in size as they sprout nerve processes, which, grouped into plexiform layers, add to the thickness of the retina. During the later stages a gelatinous secretion, supposed to come from cells of both retina and lens, fills the interior with vitreous humour, thereby GROWTH 285 progressively distending the eyeball. In addition, blood vessels and other mesenchyme penetrate into the eye from the surroundings. 'This diversity and complexity of the component processes contributing to eye size makes the search for a single "growth-controlling" principle appear utterly unrealistic . . .' Further, it must be remembered that the growth of an organism or organ may be due to a multiplication of cells, which remain about the same size, or to an enlargement of cells which do not increase in number. The fmal number of muscle or nerve cells in a vertebrate, for instance, is probably attained fairly early in embryonic life, the growth of the organs thereafter occurring mainly, if not entirely, by increase in cell size. It is not entirely clear whether the two types of growth depend on quite different underlying synthetic processes, but there is obviously some con- siderable difference between them, so that there is no reason to expect that they would follow identical growth laws. Finally, it is possible for organs, or even entire organisms of relatively simple structure, to 'de-grow' or become smaller. Flatworms or coelen- terates may respond in this way to deprivation of food supplies. In higher animals, the regression of a tadpole's tail at the time of metamorphosis is a striking example. It is hardly to be expected that any very simple formula can fit all such cases. Attempts have been made to elaborate more complex ones. Perhaps the most valiant is that of Wetzel (1937) who set up an equation of extreme complexity containing over a dozen different constants, each of which was designed to deal with one or other of the factors which he supposed to be involved in the growth of a heterogeneous collection of tissues, such as an embryo. The formula was so complex, and therefore so flexible, that it could have been made to fit almost any set of data. Its justification would in fact have to be sought not in the accuracy with which it could be fitted to observations of growth, but in the experimental justification of the various parts of the formula which were concerned with the postulated underlying processes. We still know far too little about the unit processes which go to build up the overall growth rate of an embryo for such an experimental justification to be provided. Most authors recently have been content to accept the situation that the growth of a complex organism cannot either be formulated adequately in terms of any simple, global hypothesis, such as those listed above, nor can it as yet be adequately analysed into a series of part processes; whence it follows that we have to use the various growth formulae merely as convenient means of summarising the empirical observations without attempting to attribute any profound meaning to them. For further 286 PRINCIPLES OF EMBRYOLOGY progress in the study of growth as a precisely defined concept we shall have to look to investigations on the synthesis of definable and isolatable pro- teins, such as those of Spiegelman, Monod and others, on the rate of formation of adaptive enzymes (pp. 400, 409), Meanwhile, empirical studies on the growth of the organism as a whole present many points of interest which, however, there will not be space to follow in any detail here. One most interesting and technologically important aspect of the matter is in connection with the interaction between environmental and genetic factors in the determination of growth rate and absolute size. Very little indeed is known about the physiology of such process in animals, and they present a promising field for investigation. Some work of potentially fundamental importance is being made by the use of identical twin cattle. The members of such pairs of twins have exactly the same hereditary constitution. Bonnier, Hansson and Skjervold (1948) have shown that their growth rates, although considerably influenced by the genes, can be modified to quite an important extent by the level of feeding during the growing period, but that two identical twins, one reared with abundant nutrition and the other with much poorer supplies, will even- tually tend to reach about the same final adult size although approaching this at different rates. Again, King (1954) fmds that if a twin, is kept for a period on a low-level diet and then changed to a high level, it soon makes up for any stunting it may have undergone, and proceeds to grow at the fast level characteristic of its abundant nutrition. After a certain period on the high diet, it will be heavier than its co-twin if the latter has been given the same diets in the reverse order, first high and then low. The mechanisms controlling fmal size, i.e. the factors which cause an animal eventually to stop growing, are scarcely understood at all. Some species probably never cease growth; this is said to be the case for fish. Others stop at a certain size, although the tissues are still capable of grow- ing, and will do so if a part of the body is amputated. Others again (e.g. mammals) grow till they reach a certain age, and, as we have just seen, tend to reach a characteristic limiting size in the growing period. There may, perhaps, be no general mechanism which operates in all these different situations; if there is, it is still obscure. Moment (1953) has suggested that the limit might be set by the gradual building up of differ- ences in electrical potential. Since tissues consist of cells, which are semi- permeable bags containing electrolytes among which active chemical changes are going on, it is to be expected that potential differences will exist; and they have in fact been detected (Review: Lund 1947). Moment GROWTH 287 supposes that the extremities of an animal tend to become electro- positive and thus favourable to oxidations, and this, he argues, is inimical to growth. The evidence for the existence of such a situation (or for its effectiveness if it does exist) is not very strong. Perhaps a more plausible mechanism is to be found in auto-inhibitory effects, of an immunological nature, such as those postulated by Rose and others (p. 193). Another aspect of the overall growth rate is its dependence on endocrine secretions, particularly those of the pituitary. There is not space here to deal with this subject, which belongs to endocrinology rather than embry- ology. 2. The relative growth of parts It is obvious that the different parts of an embryo do not always grow at the same rate. Several different lines of attack on the problem have been followed. Perhaps the simplest is that opened up by Huxley (summarised Huxley 1932, see also Medawar and Clark 1945, Symp. Soc. exp. Biol. 1948, Zuckerman 1950). He showed that if x is the magnitude of a whole organism and y that of some part of it, the relation between them can often be represented by the equation y = bx'^ or, what is the same thing, log y = log b -{- a log x. As the second equation shows, the two magnitudes will give a straight line when their logs are plotted against one another (Fig. 13.3a). There is no doubt that the formula does fit rather well to very many sets of data and is a very useful generalisation. The phenomena has passed under a variety of names, of which heterogony and allometry seem to be the most usual. It is not at all easy to decide just what the formula means in biological terms. Taking it from the simplest point of view, we may say that Hs a relatively unimportant constant, which specifies the size of the organ y when the whole organism x is unity. The other constant a is the one which relates to the rate of growth of the organ: if the growth rate of both x and y is proportional to their actual size we shall have dx dy -J- =Ax, / = Bx, dt dt whence y = bx^'"^, or y = bx^, when a = B/A. 288 PRINCIPLES OF EMBRYOLOGY But if we adopt a more realistic formulation of the growth rates of x doc and y, making — =f{x, t), when /(a:, () is one of the functions discussed at in the previous section, it can be shown that although the allometry formula is often a good approximation, it will only be exactly true in exceptional cases. There are other reasons why the formula cannot be accepted as a strictly accurate description of the situation. The most important is that if two 2 dist. segments Log wt. carpus Figure 13.3 A, the log weight of the two distal segments (crosses) and the two proximal segments (circles) plotted against the log weight of the middle segment (the carpus) of the claw of the Fiddler crab Uca pugnax. The slope of these lines defines the allometric growth constants B, the gradient in growth constants along the claw in Uca (full line) and the spider crab Maia (dotted line). (After Huxley 1932.) segments of an organ, y^ and y2, are each related heterogonically to the whole organism, then the sum of the two segments cannot be so related, since if jx = ^i^:"^ and ji = ^2^"^ then y-^ + 72 cannot be of the form hx"^, though the discrepancy is usually not very large. One must conclude that the allometry formula, like the other growth equations discussed previously, can at best be taken as a useful empirical summary of a set of data, but that it is not a firmly based theoretical prin- ciple. Even with this limitation, a number of conclusions can be drawn from it. In the first place, as long as a remains constant, the rates of growth of the two parts are preserving a constant relation to one another; and it is GROWTH 289 a remarkable fact that they so often do so. One simple physiological explanation of such a system would be the hypothesis that the organs are each competing, with constant efficiencies per unit mass, for a generally available supply of nutrients. An attempt to test this has been made by Twitty and Wagtcndonk (1940), who transplanted eyes of various ages and sizes between different individuals of axolotl, which were fed at differ- ent levels. They found that the simple scheme of constant efficiencies of competition was certainly not the whole story, since, for instance, a young eye transplanted to an older host which was starved might continue to grow even when the host was declining in weight. They concluded that the assimilative capacity of an organ must change (usually if not always decreasing) during the course of development (cf. p. 298). A similar conclusion emerges when one studies the growth of fragments of an organ isolated in vitro : pieces of chick heart, explanted from embryos of increasing age, show an increasing lag period before they start growing and a decreasing rate of growth in the first few days, though these differ- ences fairly soon disappear (Medawar 1940). Several authors, beginning with Teissier in 193 1 and Needham in 1932, have applied the allometry formula to the increase in various chemical entities during development (reviewed in Needham 1942). If wet weight is plotted on double-log paper against dry weight, or glycogen, fat, pro- tein, ash, calcium, phosphorus, etc., plotted against each other or against wet or dry weight, a series of straight lines are obtained. This indicates that the entities are related in the manner of the allometry equation log X = a log y + constant. The very interesting fact emerged that if we measure a number of differ- ent substances, x, y, z, etc., in two different animals A and B, we find relations of the kind log J =alog^=^log^, ^1 ^2 -^3 -Dl -D2 -D3 in which A^, Bi, etc. are different constants, but a, j3, remaui the same, whatever the animals in which x and y are measured. There is thus the same general relationship between a particular x and y (say fat and glycogen) throughout the animal kingdom or a great part of it. Needham spoke of tliis as a 'chemical ground-plan of animal growth'. Wadduigton (1933c) suggested that one might envisage the situation in terms of a general speeding up or slowing down of a basic chemical 290 PRINCIPLES OF EMBRYOLOGY programme. We can express the growth rates of entity x in animals A and B as two time-functions: Ai ±Ji Now we could choose another unit for measuring time, f^, such that X fa{t^) = fhif)- This would amount to measuring the growth rate of— ^ in time-units which made it identical with that of — . The important point is that this same transformation of the time-unit would automatically convert the growth rate of — into that of— , and that of -j- into that of ^ A2 -B2 A2 -B3 etc. Thus one change in time-scale would convert the whole chemical growth system of one animal into that of another (apart from the compli- cation due to the constants Ai, A^, etc. which express the initial state of the system when growth starts). We have here an approach to a concept of 'biological time', by which is meant the notion that events in, say, a mouse or an elephant, are similar but are all uniformly speeded up in the former as compared with the latter. It is not yet clear to what sort of entities such a notion can be applied : for instance it seems most improb- able that any such relation can hold for molecular enzymatic processes. Further discussions of it will be found in Brody (1937) and du Noiiy (1936) and some highly critical remarks in Medawar (1945). 3. Growth gradients and transformations of shape In a complex organ, it is often found that the growth rate, relative to some standard part, varies in a graded manner from place to place. The simplest expression of this can be seen when there is a series of more or less comparable parts, for each of which an allometric growth constant {a) can be ascertained. Huxley (1932, Reeve and Huxley 1945) has de- scribed many examples, relating for instance to the joints within a crus- tacean limb, or the series of limbs attached to the different segments of the body. Fig. 13.3!^ shows how the a\ for the different segments fall into an orderly sequence, which can be taken as defining a growth gradient. If one measures the growth constants for a series of distinct sub-units within an organ such as a limb, there are of course defmite jumps in its value between adjacent segments, and what should, perhaps, be a continu- ous gradient, is described in terms of a discontinuous series of steps. Examination of other cases indicates that in fact the gradients within a single mass of tissue are usually, if not always, continuous in gradation. GROWTH 291 One method of illustrating this was introduced by D'Arcy Thompson (1916), although he used it to compare adult forms which are evolution- arily related rather than ontogenetic stages of a single individual. He took one adult form as a standard, drew it in outline as seen when projected on to a plane, and superposed on the drawing a rectangular network. He showed that if this network is treated as a grid of co-ordinates, and is then distorted in the appropriate manner, the drawing of the original shape will be distorted at the same time into a fairly good outline of some other type of animal (Fig. 13.4). Such distortions of a co-ordinate network will produce alterations which are continuously gradated over the whole area, and thus the growth gradients which are affected are probably also continuously graded, since they are likely to depend on the same funda- mental mechanisms as produce the distortions which differentiate one form from the other. More recent workers have in fact shown that the same method can be used to compare different developmental stages of a single species. (^ .< ~~*""^ l^p c 0 ' fl "S R \, ^ _^ — ■ ^ ^ 0 12 3 4 5 6 Figure 13.4 A transformation of a co-ordinate grid which converts the outline ofDtodon (left) into that of its relative the sunfish Orthagoriscus. (After D'Arcy Thomp- son 1942.) 292 PRINCIPLES OF EMBRYOLOGY D' Arcy Thompson's suggestions opened up a large field for investigation, but unfortunately this has not been as systematically studied as might have been hoped. We can deal v^ith the recent work under three heads : firstly, improvements in the method; secondly, the general physiology of growth gradients and shape transformations, and thirdly, attempts to discover the physiological mechanisms underlying them. Actually rather little has been done to make D'Arcy Thomson's method of the distortion of a co-ordinate network into a means of exact analysis. Medawar (1944, 1945) has made some steps in this direction (see also Richards and Kavanaugh 1945). He considered the changing shape of the human body during its development from the early foetus to the adult. The body was first reduced to a two-dimensional shape by repre- senting it as a series of outline drawings when seen from the front (Fig. 13.5). It becomes obvious then that in the early stages the legs grow faster than the parts nearer the head, and it appears probable that there is a single continuous growth gradient with its high point towards the feet, falling off as one goes higher up the body. Medawar pointed out that this could be represented by a transformation of co-ordinates and that this trans- formation could theoretically be specified in algebraic terms. To illustrate how this might be done he reduced the shape of the whole living body still more drastically and considered only certain points on the vertical midline; the foot, fork, navel, nipples, chin, etc. The original three- dimensional shape was thus reduced to a line on which certain intervals are marked. Suppose now that Pi, P2, P3, etc. are the heights from foot to fork at successive points in time, and similarly Qi, Q2, Q3, etc. the heights from fork to navel at the same times, and so on for the other intervals. We can from the actual measurements work out empirical equations connecting the successive Ps and again, another set of equations connecting the successive Qs, and so on. Each equation will give the changes of one part of the network as time proceeds. We can also fmd algebraical relations between the equations relating to the Ps and those relating to the Qs, the Rs and 5s, etc. at any given point in time. With the aid of these two sets of equations, the whole series of transformations can be expressed algebraically. It is clear, however, that quite a lot of arith- metic is required to produce even a fairly clumsy algebraical description of a series of shapes, notwithstanding that these have been reduced to their very simplest form, the original three dimensions having been whittled away to one. Such labour is only justified if it enables one to see certain relations which would otherwise be missed. So far, such evidence of a real usefulness of the method has not been forthcoming. There are contexts, however, in which it seems probable that Medawar s GROWTH 293 methods would be useful if they were applied. The changing shape of animals as they mature is of practical importance in relation to the production of hvestock. Hammond (1950) has been particularly con- cerned with the problem and has demonstrated a number of general points about the physiology of growth gradients. These he has been able to illustrate pictorially, but it has so far not been easy to reduce them to a precise and manageable form; Medawar's methods might be very useful in this connection. 2575 Years Figure 13.5 Changes in the proportions of the human body during growth. The heights of certain landmarks (knees, fork, navel, mouth, etc.) were ascertained for each age, and related to one another by means of empirical equations. From these equations the heights were recalculated, to give the horizontal lines which are drawn on the figures. The goodness of fit of these lines indicates the degree to which the changes in proportions have occurred in a regularly graded manner, so as to lend themselves to summarising in relatively simple algebraic functions. (From Medawar 1945.) Hammond shows that the various species of wild animals from wliich our domestic livestock have been derived have each their characteristic pattern of changes in shape during development. These can be illustrated rather vividly if one takes as a standard an early developing part such as the head and shows a series of drawings or photographs of different stages of the animal, all of which have been adjusted to the same head size. We then see that in the horse, for instance (Fig. 13.6), up to the time 294 PRINCIPLES OF EMBRYOLOGY of birth there is a great decrease in the length of leg relative to the re- mainder of the body, whereas after birth the most important changes are a lengthening and deepening of the body. In wild sheep the changes are somewhat similar, though perhaps not so marked. In wild pigs there are no very marked changes in proportions from the foetus up to the adult. It is these basic developmental patterns which are altered either by the genes selected by the livestock breeder or by the conditions of feeding Figure 13.6 Changes of proportion in the growing horse. The upper row of drawings show a primitive type of horse at various stages from the foetus to the adult, adjusted in size so as to have the same length of cranium. By the stage of the first drawing (late foetus) there has already been a great development of the legs; later there is a preponderant growth of the trunk. The two drawings below show on the left a thoroughbred, and on the right a draught-horse ; the later phase of growth has been minimised in the former and enhanced in the latter. (After Hammond I950-) and husbandry under which he keeps his animals. Among horses, for instances, the strains bred for speed have been produced by selecting genes which partially suppress the later changes, so that the adult horse retains the long legs and slim body of the normal juvenile phases. In the heavy draught horses, on the other hand, genes have been selected which increase these later changes so that one obtains an animal with a very large and heavy body and relatively shorter legs. In the mutton breeds of GROWTH 295 sheep it is again genes which encourage the later changes in body confor- mation which are required and in the improvement of pigs it is also the late-developing hindquarters rather than the early-developing head and forequarters that it is necessary to emphasise. The changes in body proportions can be affected not only by genes but also by the level of nutrition on which the animals are kept. For instance, if two comparable sets of pigs are kept, one on a high plane of nutrition and another on a low plane, until they both reach the same weight, it will not only be found that the high-plane pigs reach the speci- fied weight more quickly, but that the two sets of animals differ in con- formation at the end of the experiment. The low-plane animals retain a more juvenile shape. One can say that in spite of maturing more slowly they mature in a conformation which is characteristic of a younger animal than do the similar pigs reared on the high plane of nutrition. Hammond explains the situation in the following ways: The different regions of the body, such as the head and the forequarters, the hindquarters, etc., attain their greatest rate of relative growth in succession. The same is true of different tissues, such as bone, muscle, fat, etc. The funda- mental sequence in which these various parts come to the fore is never altered, but changes in genes or changes in nutrition can either compress the sequence into a shorter length of time or spread it out over a longer interval. High nutrition brings the successive phases nearer together in time; so do the genes which determine the draught type of horses or the meat-producing type of sheep or cow. Low nutrition spreads the phases further apart and so do the genes for racehorses (Fig. 13.7). It appears from other experiments of Hammond and his associates that even if the plane of nutrition restricts the outward expression of the sequence of phases the basic physiological changes determining them may be proceeding nevertheless. Thus two sets of pigs were brought to the same weight in the same length of time, but by different routes ; one being kept first on the high plane and later on a low, the other first on a low and later on a high plane of nutrition. They nevertheless showed characteristic differences in conformation. There is obviously a great deal more work to be done on the physiology and the genetics of these rela- tions. It is one of the most fascinating, and also most practically important, aspects of the whole subject of growth. Our knowledge of the mechanisms which control growth patterns is very meagre. We have to recognise in the first place that the processes which finally issue in an adult shape may be very complex. Waddington (1950^7) has discussed one particular case from this point of view, that of the wings o£ Drosophila and shown how the fmal shape depends not only 296 PRINCIPLES OF EMBRYOLOGY on successive phases of cell division and cell expansion but also on defor- mations of the whole structure resulting from the changes in pressure of the body fluid contained in it. No very simple physiological account of the general growth pattern can be expected in such complex cases. Even when we are dealing with a simpler case, in which the dominant factor is growth in the ordinary sense of cell multiplication and cell enlargement, our present knowledge does not provide a basis from which the phenomena can be easily understood. The growth pattern seems to Figure 13.7 Diagrammatic curves illustrating the succession of anatomical systems v/hich are predominant in growth rate in mammals (particularly farm live- stock). Curve I relates to the cranium and shanks, bone and gut-fat, which have a high growth rate at an early stage; the neck, main body musculature and subcutaneous fat grow fastest at a rather later stage (curve 2), and the hind-quarters and intra-muscular fat still later (curve 3). The upper set of curves show the situation when the animal is kept on a high plane of nutri- tion; the phases follow one another rapidly. Under conditions of poor nutrition (lower curves) the succession is more long dravm out. (After Hammond I950-) characterise whole regions, which may be made up of a number of anato- mically different elements, and it is surprising how often these elements appear to work together in a harmonious way. Fig. 13.8 shows in profile the skulls of a number of different types of dogs. The differences are pre- sumably genetically determined and it is clear therefore that genes affect the pattern as a whole and not only the individual units comprising it. Moreover it must be remembered that each skull is made up of a number of different bones; to mention only the most striking example, the lower GROWTH 297 jaws are usually modified in a way consistent with the shape of the upper part of the skull to which they are attached. This subordination of indivi- dual parts to the whole to which they belong is the general rule, but it is not quite universal. For instance, in the Fg and later generations of certain crosses between different breeds of dogs, some cases of definite disharmony between upper and lower jaws and between other parts of the body may be found, but they are relatively rare. Co-ordination usually extends not only between parts of the same general nature, such as bones, but affects tissues of quite a different kind, such as skin, muscle, etc. It is, however, rather commoner to find instances in which the growth rates of markedly different tissues are not properly assimilated to one another. For instance, in dogs with greatly shortened faces, such as bulldogs, the skin is often too large for the bony structure and therefore has to hang in folds (Stockard 1941). Figure 13.8 Skulls of various races of dogs, adjusted to the same size, to illustrate how the structure, although complex, is modified as a whole, (a) German sheep dog; (b) Borsoi; (c) German Dogge; (d) St. Bernard; (e) Zwergspitz; (j) Itahan Windspiel; (g) Englischer Mops; (/i) Japanese spaniel. (After Huber 1948.) Very little is known about the ways in which such growth correlations arise. General endocrine control affecting all types of tissue certainly plays a part in many cases. It seems probable also that the growth rate of one organ or part of an animal usually has some influence on the growth rate of the neighbouring regions. For instance, Huxley (1932) compared the growth rate of the limbs in Crustacea in species in which one sex has a very large, fast-growing hmb, which is absent in the other. He showed that the presence of a fast-growing limb tended to affect its neighbours, in 298 PRINCIPLES OF EMBRYOLOGY general increasing the growth of the limbs immediately posterior to it and depressing the rate of the Hmbs immediately in front. The mechanism of the effect— whether the fast-growing limb operates by secreting some growth-promoting substance or by competing in some way for a limited supply of available raw materials— is quite unknown. One of the most extended series of studies of the physiology of growth rate of individual organs is that by Harrison, Twitty and others on the eyes and other organs of the Mexican salamander Amhly stoma (Reviews: Harrison 1933, Twitty I934, Needham 1942, Reeve and Huxley 1945). The eye of one species, A. tigrinum (the normal axolotl) is very much faster growing than that of the nearly related species A. punctatum. It was first shown that if the eye-cups are interchanged and transplanted from punctatum to tigrinum or vice versa, each type retains its own characteristic growth rate even in the new situation. The growth rates are therefore inherent in the eye-cups themselves. When organs are grafted into other animals of the same species but different age, the growth rate of a transplant younger than the host is speeded up, and that of an older one slowed down, until the grafted structures have reached a size appropriate to the body in which they lie. One hypothesis to account for this would be that the young organs have a greater assimilative efficiency than older ones, and are therefore able to obtain more than their normal share of nutrients from the blood stream when they have only older organs to compete with. But that can hardly be the whole story. Even when the competitive demand for nutrients was m- creased as much as possible (by starvation, to such a point that the host body lost weight, together with amputation of the tail which caused this to regenerate) the young transplanted eyes were still able to grow. Twitty came to the conclusion that at least two factors must be involved; not only a decreasing assimilative efficiency of an organ as it ages, but also an increase with age of the richness of the nutritive suppHes in the blood (Twitty and Wagtendonk 1940). These ideas and observa- tions are obviously closely related to those of Hammond mentioned above. In other experiments by Twitty, the two main elements of the eye— the eye-cup and the lens— were combined in different ways. Here there was definite evidence of the influence of the growth rate of one element on that of the other. For instance, if a large eye-cup of Amhlystoma tigrinum is combined with the small lens of ^. punctatum or is allowed to induce a lens in punctatum skin, we find that in the compound eye, the eye-cup of tigrinum grows more slowly than usual while the lens of punctatum grows more rapidly, so that the two finally come to bear a GROWTH 299 harmonious relation to one another. The same kind of thing is true in the opposite combination of a small punctatum eye-cup with a large tigrinum lens; the growth-rate of the eye-cup is increased and that of the lens decreased until the two fit. Again the mechanism of the effect is quite obscure. Weiss (i 949^2) has suggested that it may be a matter of tension exerted on the growing edge of the retina. In a combination where the lens is too small, it will permit some of the vitreous humour to flow out of the eye-cup and thus the tension will be reduced and the growth rate lowered. On the other hand, an over-large lens will exert a radial pressure against the edges of the retina and this might lead to an increased growth rate (Figs. 13.9, 13.10). Figure 13.9 Reciprocal effects of lens and eye-cup. Figures a and h show the eyes of a fairly young tadpole of Tritums tacniatiis in which the lens of the left eye {a) has been induced out of axolotl ectoderm grafted into the region; the lens is much too large in relation to" the eye-cup. Figures c and d show a later stage from a similar experiment (at a lower magnification) ; the lens derived from axolotl tissue (in c) is still larger than the corresponding taeniatus lens, but it has caused its associated eye-cup to grow faster than normal, so that the relative sizes are nearly adjusted to one another. ("After Rotmann 1939) 300 PRINCIPLES OF EMBRYOLOGY Many other combinations between tissues from different species of Amphibia have been made experimentally. They often exert influences on one another's growth but these differences are not always mutual. Rotmann (1933) for instance, found that if on the body of a newt of the species Triton taeniatus a limb was provided with a core of mesoderm of the 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 Days after operation Figure 13.10 Relative growth of eye-cup and lens in inter-specific grafts in Amblystoma. Curve A, growth of tigrinum lens whefi associated with tigrinum eye-cup; curve B, growth of tigrinum lens when associated with punctatum eye-cup. The size of the lens is expressed as the ratio tigrinum lens to punctatum lens of same age. Note that the tigrinum lens in its own eye-cup reaches about i'6o times the punctatum size, but when combined with a. punctatum eye-cup only about 1-30 times. Curve C shows the growth o£ a. punctatum eye-cup provided with a tigrinum lens, the 'size index' being its ratio to a normal punctatum eye-cup. Note that it becomes larger than usual, to about the extent required to fit the associated tigrinum lens. (After data of Harrison.) species T. cristatus, but enclosed in skin belonging to the host's species, the form of the limb up to the time of metamorphosis was entirely dic- tated by the species that contributed the mesoderm, which imposed its own growth rate on the ectoderm without being in any way influenced by it (Fig. 13. 11). Interesting also in this respect are the experiments of Baltzer (see 1952^) who, however, was concerned not only with the growth rate in organs compounded out of combinations of tissues from two different species, but even more with the original induction of the organs and the laying down of their basic structure. On the whole he GROWTH 301 found that mesoderm was more likely than ectoderm to react to in- fluences from its surroundings and become assimilated into a compound organ. Figure 13. ii The two forelegs of a newt, T. taeniattts, in one of which (the lower in the figure) the mesoderm of the limb-bud had been replaced with tissue from T. cristatus. The operated leg has developed the long, rapidly growing toes typical o£ cristatus. (From Rotmann 1933.) SUGGESTED READING Medawar 1945, Reeve and Huxley 1945, Weiss I949i is greater than b^, Ri will be larger than R2» and there will be dominance of the site with greater efficiency over that with less. It is clear, without our going into the details of the other possible cases (see Spiegelman 1945), that the assumption of physiological competition does provide a mechanism by which gradients in efficiencies of synthesis or interaction would give rise to phenomena such as dominance. It thus makes it possible to envisage field phenomena in a form in which they become amenable to physiological analysis, aimed for instance at 314 PRINCIPLES OF EMBRYOLOGY measuring the relevant efficiencies or discovering what substances are being competed for, etc. As to the 'substances' involved (we have to use the word in a broad sense), we have two main pieces of information, which have not yet been fully brought into relation with each other. The first to be discovered was that oxygen is highly important. It probably operates in two ways, firstly as a component of the stimulus which sets the regeneration going, and secondly as one of the reactants while the process is proceeding (Barth 1940). Its importance as a stimulus can be demonstrated if a hydranth is cut off and the perisarc pulled forward and ligatured in front of the cut surface, so as to shut it off from the surrounding sea water; no regenera- tion takes place. The same result can be obtained by covering the cut surface with a piece of glass tube. Moreover, if the perisarc is cut open so as to expose a region of the coenosarc, regeneration may occur, particu- larly in oxygen-rich water, even if no wound has been infhcted; and the injection of a bubble of oxygen between the perisarc and coenosarc may have the same effect. The continuing importance of oxygen during the whole process of regeneration is shown by the fact that the rate of for- mation of new hydranth is highly dependent on the amount of oxygen in solution in the water. Child, and many workers following his lead, have been emphasising for some time the importance of gradients of respiratory rate which they claimed to demonstrate in hydroids and many other regenerating animals, and in eggs in which field phenomena play a leading role (see Child 1941). It was claimed that field processes always depend on gradients in metaboHc activity, and that the metabolic activity which is most crucial is that of respiration. The expression 'metabolic activity' is, of course, so general that, in those terms, the hypothesis is little more than a truism; a field must obviously have graded differences between its parts, and the port- manteau phrase 'metabohc activity' could cover these whatever their nature. The part of the theory which it is important to discuss is, therefore, the notion that it is respiration which is basic. Extended discussions will be found in Child's own book (pro) and in Needham 193 1 (pp. 582 ff.) and Brachet 1945 (contra). The general sense of the situation would seem to be that, whereas various indirect methods (e.g. susceptibiHty to poisons, reaction with vital dyes, etc.) often give evidence for the existence of some sort of gradient, it is by no means clear in most cases that the gradients primarily affect respiration; and moreover it remains obscure whether the gradient of respiration, if there is one, is the causative basis of the observed field or rather merely another expression of it. The two most crucial embryological cases in which critical evidence might be REGENERATION 3I5 sought are those of the echinoderm animal-vegetative gradients and the amphibian organiser; in neither of them is a gradient in respiratory rate of demonstrable importance (see pp. 88, 200). In the hydroids, the situation is somewhat more favourable to Child's ideas, since there is fairly good evidence that a gradient in rate of respira- tion actually exists, hi the early measurements of the oxygen uptake of fragments taken at successive levels behind the hydranth, the experiments lasted so long that an important amount of regeneration would have occurred, and the gradient of rate found, with the hydranth end respirhig fastest, may have been mainly an expression of the faster rate of regenera- tion of this end. But Barth (see 1940) has made accurate measurements over short periods, and finds that there is almost certainly a gradient in this sense in the hydroid immediately after cutting. This, however, does not by any means necessarily imply that the respiratory gradient is a cause, and not a mere concomitant of the gradient in regeneration rate. Turnijig now to a type of 'substance' quite other than oxygen, it seems that considerable importance should be attached to the fmding of Tardent (1954) that there is a gradient in the concentration of neoblasts or regenera- tion cells (known also as interstitial cells). This was found both in Hydra and Tubular ia (Fig. 14.3). These cells increase in number near the cut surface, but it is not yet clear to what extent this is due simply to multiplication or how far migration from other parts of the animal is involved. If the latter process is extensive, it may be that they are the most important 'substance' (if one can call them that) for which the two ends of an isolated length of hydroid are competing. It would be very interesting to know whether their rate of respiration is liigher than that of the other tissue cells, in which case the respiratory gradient described by Child and Barth might be a reflection of the gradient in neoblasts ; but there is as yet no defmite evidence on this point. It seems probable that when a hydranth is removed from a hydroid, the gradient from the new distal end is established in two stages. First there must be an accumulation of neoblasts at the wound surface, pre- sumably as a response to the increased availability of oxygen. But after the initiation of the hypostome or mouth region of the new hydranth, this may be itself responsible for building up the gradient. Several authors have shown that this region, when transplanted into the side of another polyp, is particularly efficient at causing the production of a new hydranth in its neighbourhood, much of which is formed out of tissues of the host (Beadle and Booth 1938, Yao 1945). This is a typical example of an 'assimilative induction', in which the transplant acts as the carrier of a powerful individuation field. In Hydra, whose powers of regeneration 3l6 PRINCIPLES OF EMBRYOLOGY are more moderate, only the hypostome can induce a new hydranth in this way. hi Tubularia, on the other hand, as we have seen, mere lacera- tion, or the injection of a bubble of oxygen, will suffice to produce the same result, hi such extremely regenerative forms, the tissues can be cut up fmely and reduced to a homogeneous mass, and are still capable of giving rise to a well-organised hydranth from the upper surface where the availability of oxygen is greatest (cf. Barth 1940). Figure 14.3 Frequency of interstitial cells along the stem o£ Tubularia. (After Tardent 1952.) It is worth noting, as a curious and so far unexplained fact, that a hy- dranth which is actively in the process of regeneration is not able to re- store itself if injured before the process is complete (Davidson and Berrill 1948). The regenerative capacity appears rather suddenly just when the new hydranth reaches its final functional histological state; and it will then not only react by regeneration to any subsequent injury, but will replace parts which have been removed during its development. This may perhaps mean that during the first regeneration all the neo- blasts are involved in the process and there are no more available to deal with a second injury, but the matter has not been fully investigated. It is REGENERATION 3I7 commonly found that embryos in the most active phases of development have less power of regeneration than at either earlier or later more quiescent periods. Thus in the Amphibia the early gastrula can easily repair defects, and so can the young tadpole, but the neurula or early taU-bud stage has little capacity for regulation. {b) Flatworms It will be useful to supplement the account of regeneration fields in hydroids by a somewhat shorter discussion of similar phenomena in flat worms, which are also a lowly group of invertebrates but somewhat more highly evolved than the coelenterates (General Reviews: B rousted 1954^, b, Wolff 1953). We shall fmd that in the flatworms the active individuation field in the adult is less powerful than that wliich is main- tained by the hydranths in hydroids. As a consequence of this the main role in directing the early course of regeneration is played not by the adult organs which remain in the regenerating piece, but rather by a static field of regeneration potential, which can be compared with the gradient in regeneration rate which was characteristic of the hydroid stem. This determines the character of the blastema which is formed, and that in its turn then brings about the development of the appropriate organs. Regeneration has been mainly studied in triclads, of which Planaria is a characteristic genus. The whole group are frequently referred to as planarians. If a planarian is cut in two by a transverse cut it is frequently found that the anterior segment regenerates a tail and the posterior seg- ment a head. In both cases the process starts by the formation of a blastema, which is produced by neoblasts which migrate to the wound surface (see p. 306). Many species of planarians are provided with eyes in the anterior region of the head. One of the first signs of head regeneration is the ap- pearance of such eyes in the blastema. This occurs at an early stage, even if a large part of the anterior of the worm has been removed. Thus it seems that in regeneration of the head the most anterior part is formed first and whatever else is required is, as it were, intercalated between this anterior part of the head and the remaining posterior end of the body. During the later stages of regeneration, however, a good deal of morphal- laxis occurs, that is to say a remodelling of the original posterior part of the body. The occurrence of these easily recognised organs, such as eyes, makes it simpler to study the regeneration of the head than that of the tail, and most work has concerned itself with this type of regeneration. The sim- plest experiment consists in placing the transverse cut, which divides the worm in two, at various levels from the anterior to the posterior. We fmd 3l8 PRINCIPLES OF EMBRYOLOGY that, as was the case in the hydroids, any particular level of the body may form either a head or a tail according to whether it is attached to an posterior or to a anterior piece. However, the ability to regenerate a head, as measured either by the frequency with wliich this is successfully accom- plished or by the time taken to do so, is not usually constant along the whole length of the worm. Different species fall into a number of classes in this respect. There are some in which no regeneration of a head occurs from any part; in others, of wliich the well-known Dendrocoelum lacteum is one, the anterior end regenerates easily but the abihty to form a head falls off rapidly and has disappeared at the level of the pharynx. In others, the curve expressing the ability to regenerate a head (the so-called 'head- frequency curve') falls off more gradually and even the most posterior end of the body sometimes carries out the regeneration successfully. Some Planaria normally reproduce by transverse fission, a new animal forming in the posterior end of the body of an old one. In these the head-frequency curve, after falling in the region of the pharynx, rises again towards the posterior end. Finally there are some species, such as Planaria velata, in which the ability to regenerate a head appears to be equal along the whole length of the body. There are also tail-frequency curves in all these species, but less is known about them. The ability to regenerate a head is, however, not fully expressed merely by a curve which assigns some defmite ability to each body level. There is, in point of fact, a gradient from the midline of the body towards the margins as well as from anterior to posterior (Fig. 14.4). We have to deal with a two-dimensional field of head-forming ability rather than with a one-dimensional gradient of it. The type of regeneration which occurs when the planarian body is cut in more complicated ways can usually be deduced fairly simply from the pruiciple that lq posterior pieces a head forms at that point of the cut surface wliich has the highest value in the head-producing field. To account for the fact that the same point would form a tail if attached to the anterior piece, the simplest assumption seems to be that the regeneration tends to occur in such a way as to carry on the gradient which is already intrinsically present within the fragment. In this way one can understand such peculiar phenomena as the appear- ance of the tw^o heads at the shoulders of the T-shaped cut shown in Fig. 14.5A-D, The appearance of heads in the situation shown in Figs. 14.5E and F is not so fully accounted for, since here the regenerating edge is connected directly both with the anterior and the posterior parts of the body, and further subsidiary hypotheses would be necessary before it was clear what we might expect to obtain. There seems at present to be no adequately tested hypothesis which can deal satisfactorily with all the REGENERATION 319 numerous and complicated experiments which have been made in this field. It is important to note that it is only regions of the old body in the im- mediate neighbourhood of the cut which exert an influence on the course of regeneration. Bronsted (1939) has isolated a section of the body anterior to the pharynx by two transverse cuts (in Euplanaria luguhris) and then grafted the anterior tip containing the original head with reversed polarity to the posterior edge of the isolated segment. The grafted head in this Figure 14.4 On the left, the head-regeneration field in the planarian BdcUocephala punctata. On the right, a head regenerated in a 'window' cut in the anterior region. (From Bronsted 1946.) situation exerted no apparent influence on the regeneration at the anterior- cut surface, which proceeded to form a head exactly as it would have done normally. Similarly, if a window is cut in a planarian, as in Fig. 14.4, the existing old head does not inhibit the regeneration of the new head in the window. Again, Raven and Mighorst (1948) have shown that the rate of head formation at a given level in Euplanaria lugubris is not in the slightest affected by the presence or absence of a further posterior cut at which, if it is present, a tail will simultaneously be regenerating. Thus in planarians there is little or no evidence of competitive interaction between the two ends of an isolated segment, or between a regenerating surface and 320 PRINCIPLES OF EMBRYOLOGY already existing organs. We have thus httle sign of anything correspond- ing to the phenomenon of dominance in hydroid regeneration. There are, however, some inductive interactions in operation m plan- arian regeneration. If the head region oi Dugesia lugubris is grafted into the posterior of the body, behind the pharynx, it can mduce the formation of a supernumerary pharyngeal region from its neighbourhood; while in PolyceUs nigra, the cerebral ganghon induces the formation of eyes Figure 14.5 AB C D, staaes in the regeneration of two heads, following the T-shaped cut sho-^ in A; E, F, regeneration of a head following a longitudinal cut. (After Beissenhirtz, from Br0nsted 1946.) though only from a limited competent area which extends from the anterior to the level of the pharynx (work of Sengel and Lender see review of Wolff 1953). Wolff has summarised the conclusions from these experiments in a diagram which is reproduced as Fig. 14-6. The first event m a blastema engaged in head regeneration is the formation of the ganghon; this induces a cephalic region (which in many forms includes eyes); this region induces a pre-pharyngeal region, and that again a pharyngeal region; and in the latter the pharynx itself eventually appears. These inductive relations can be regarded as the expressions of an mdividu- ation field which is most powerful when it arises within a blastema, since it can then affect even the anterior part of the body, but which persists at a lower intensity even in the fully developed organism. REGENERATION 321 Figure 14.6 Successive inductions during the regeneration of planarians. After a poster- ior region is isolated (a) the first step is the formation of a gangHon (C) in the blastema {b). This induces a head (T) provided with eyes' (y). The head region then (d) induces a pre-pharyngeal zone (Pr), and that in turn (e f) a pharyngeal zone (Ph), in which (g) a pharynx finaUy appears. (From Wolft I953-) {c) Amphibia Larval and adult urodele Amphibia (newts, salamanders, axolotls, etc.) can regenerate legs or tails and other organs fairly readily (General Re- views: J. Needham 1942, A. E. Needham 1952). The capacity of Anura (frogs, toads, etc.) is much less in this respect, the power of regeneration usually being lost at about the time of metamorphosis. The regenerative phenomena in urodeles provide a very good example of one kind of field action. It was in fact in this connection that the concept was first exten- sively discussed by Weiss, one of those who introduced the notion of 222 PRINCIPLES OF EMBRYOLOGY fields into embryology, and who contributed greatly to the early experi- mental work on vertebrate regeneration (see Weiss 1930, 1939). As we shall see however, the field which is operative in ampliibian regeneration is strictly an individuation field concerned with the buildmg up of a com- plete unit, and shows no sign of activity outside the Hmits of this unit. There is therefore nothing which corresponds to the dominance ot the old hydranth in the regeneration of hydroids. . , 1 As far as regeneration is concerned the urodele body can be considered to be made up of a series of organ-districts. If a complete district is re- moved, It caimot be regenerated. If, however, any part of the district is ehminated regeneration wiU restore what is missing, unless indeed tor some reason the blastema is smaller than normal, when a deficient organ may be produced. The limit of the organ district of the tail, for mstance, is the last sacral vertebra. If the tail is amputated anterior to this, no re- generation occurs, whereas if the cut is made anywhere further posteriorly, a complete tail is formed. Regeneration occurs strictly withm each organ district and is uninfluenced by the position of that district withm the body as a whole. Thus if a limb is transplanted to the middle of the back and then amputated, the stump nevertheless regenerates a hmb. The character of the regenerate, m fact, depends on the tissue in the immediate neigh- bourhood of the wound. If a hindUmb is amputated, a forehmb trans- planted to the stump, and then the forehmb agam amputated so as to leave only a small section of it, the regenerate will still be a forehmb and show no influence of the hindhmb stump, which is further away from the wound surface. 1 • 1 Amphibian regeneration exhibits several pecuhar polarity phenomena. In the first place it should be noted that the regenerate always produces the parts distal to the wound surface and apparently never those proximal to it Thus if a deep V-shaped cut is made into a hmb, both faces of the wound may produce a regenerate and both of these will develop into the regions of the hmb which should he distal to the cut. Agam, it a segment of a hmb, say the region near the knee joint, is isolated by two cuts and then inserted into the body-wall in such a way that the Proximal surface as well as the distal can regenerate, it will be found that the proxknal surface does not form a new femur and upper part of the hmb but that both surfaces form the distal extremity. J. Needham (1942 accepted some earlier data which suggested that in such cases the proximal surface regenerated only so much of the lower limb as had origmally been isolated. This would have been a very pecuhar situation, since it would have meant that the mdividuation field had been permanently altered by the isolation. It appears, however, from more recent work of Monroy REGENERATION 323 (1942), that the regenerate from the proximal surface forms the whole missrng region and not only a part of it (Fig. 14.7). We can conclude therefore that regeneration will always produce the whole missing region from the cut surface to the distal tip of the organ, even if this means that a reversal of polarity has to occur at the place where the cut was made It may be pointed out that if a section of a limb is removed and the distal region grafted back on to the stump, no regeneration of the missing seg- ment occurs, probably because there is no opportunity for a blastema to rorm. i^^V;^: b Figure 14.7 (Left) Part of an anterior limb of a newt, consisting of sections of the'humcr- us, and of the radius and uba, was grafted into the flank. Both ends have regenerated and each blastema has produced all the structures distal to the position of the wound from which it arose. (From Monroy 1942.) Kl^ JT ^"^.b-^,^^,f g^^^^^^es (. and b), and one {c) showiiag features of both tail and limb, all from axolotl tails onto which limb-skin has been transplanted. (From Luther 1948.) Regeneration, of the limb at least, is not only limited by the fact that it proceeds only distally and never proximally: it can also not proceed laterally That is to say, if a limb is spht longitudinally and one half re- moved, this half IS not replaced. However, if the half-limb is now ampu- tated regeneration occurs and the regenerated portion of the hmb is complete m cross-section. Similarly, if one of the long bones of a hmb is removed, it will not be regenerated, but if the hmb is now amputated 324 PRINCIPLES OF EMBRYOLOGY through the boneless region, the regenerate will be provided with the full necessary complement of bones (the rule does not hold for the tail, see page 308). This formation by the regenerate of structures which are more complete than the stump shows clearly that regeneration is not merely a result of the outgrowth of the tissues of the stump, but is a phenomenon m which an mdividuation field arises m the blastema and leads to the development of the complete section of the organ between the level of the cut and the distal tip. However, although the structure of the regenerate is not directly produced by the outgrowth of the tissues exposed at the cut, those tissues are operative in determmmg the character of the individuation field which arises. In most cases, as we have seen, a mere absence of a tissue does not lead to any deficiency m the mdividua- tion field If, however, tissues from two different organs are mixed, com- pound or hybrid individuation fields may appear Thus Liosner and Woronzowa (193?) and Monroy and Oddo (i943 W found that if muscles from the tail are grafted into the urodele limb at the site of ampu- tation the regenerate may be intermediate m structure between a tail and a limb. Similarly intermediate structures were found by Luther 1948) after the transplantation of skin from the foot to the tail (Fig. 14.7}. SUGGESTED READING For a full treatment, A. E. Needham 1952. Other recommended reading : Weiss I939. pp. 458-478; Barth 1940. Rose 1948^; Avel 1940 (oligochaetes, not treated m the text). Spiegelman 1945. PART TWO THE FUNDAMENTAL MECHANISMS OF DEVELOPMENT IN the introductory chapter a Hst was given of the basic concepts wHch experimental embryology has developed-those of determination and differentiation, brought about by the mechanisms of ooplasmic segrega- tion, evocation and field action. The accounts of the development of the various classes of animals given in Part One have exhibited the type of facts from which these concepts have been derived. The facts have, for the most part, been obtained by typically biological modes of experiment; for instance, by the transplantation from one place to another of whole cells, or even large masses of cells. The theories to which they have given rise have in consequence also been framed in terms of concepts which belong to the biological realm and cannot be immediately brought into Ime with the ideas of the more fuUy developed sciences of physics and chemistry. As Weiss (1947, 1950^) in particular has emphasised, we cannot be permanently satisfied with this situation, but must attempt to push our analyses down towards the level at which we are discussing the inter- actions, combinations and synthetic activities of particular substances. In this connection he has coined the expressive phrase molecular ecology' and there is no doubt that a fuUy developed embryology ought to be able to expound the processes of development in terms of the changes in the populations of molecules making up the ceUs of the different tissues and should not have to rely on concepts such as evocation and competence, which are special to it alone. The programme which Weiss proposes is, however, a very difficult one and we are still far from the goal which he envisages. Indeed the distance is so great that there is probably some danger in attempting to cover It m a single step. There is an intermediate level, between that of the organisers, fields, etc. usuaUy considered by embryologists and the ulti- mate molecular constituents of living matter, which requires to be thoroughly explored before we can feel the ground firm enough under out feet to have any confidence in attempting to frame theories in chemical or physical terms. This is the level which deals with the activities of the different categories of ceU constituents. As we have pointed out experimental embryology has already approached it in several diff'erent contexts; for instance, in relation to the cell granules of mosaic eggs (p. loi) the mitochondria of sea-urchins (p. 90) and the microsomes of Amphibia (p. 212). These are all constituents of die cell cytoplasm, not of the nucleus. The crucial role of inductive processes (both between tissues 327 328 THE FUNDAMENTAL MECHANISMS OF DEVELOPMENT and witliin embryonic fields) demonstrates that the initial differentials which guide the development of various parts of the egg into different channels depend in many cases on substances which can pass from cell to cell and which must therefore be extra-nuclear. There is another method of approach which has penetrated very deeply into this field of the activities of cell parts, that is the science of genetics which has operated mainly by the study of heredity. The knowledge it has yielded has in the main concerned the effectiveness of the nucleus and the chromosomes, an aspect of cell behaviour about which the methods of experimental embryology have not told us very much. More recently, moreover, genetics has begun to produce most valuable information about cyto- plasmic particles, which it knows under the name of 'plasmagenes'. It would, of course, be inappropriate to attempt to give even a sketch of all aspects of genetics in a book devoted to embryology, but there is great deal of genetical information wliich is not merely relevant but wliich is essential to our purpose. In fact, when we attempt to discuss the funda- mental mechanisms of development we are in a region in which the dis- tinction between the sciences of genetics and embryology breaks down. Genetics using its normal method ot studying heredity has revealed the existence of genes which control to a large extent the character of the animal which will develop from the fertihsed egg ; the way in which the genes operate in doing this belongs by definition to embryology. The union of the two sciences could hardly be more intimate. In the first chapter of this part we shall discuss in broad outline what genetics has revealed about the general nature of the epigenetic system and the way genes are involved in it. This supplements the experimental em- bryological material, to complete the picture of development at the bio- logical level. We shall then pass on to consider the basic mechanisms of development in terms of the activities of cell constituents, using facts some of wliich are conventionally considered to belong to genetics, others to embryology. The greater part of this discussion will be concerned with the differentiation of substance which occurs during development; that is to say with the formation of new materials or new types of tissue. The last chapter deals with the other aspect of development, the moulding of tissues into organs of definite shape and form. CHAPTER XV THE ROLE OF GENES IN THE EPIGENETIC SYSTEM THE SCIENCE of gciietics has clearly shown that when an animal differs from nearly related forms, the nature of these differences is nearly always con- trolled by genes carried on its chromosomes. It is clear then that genes must be amongst the most important causal entities which play a role in guiding development. We have so far discussed the question of why an organ, such as a limb, develops as it does in terms such as organisers, fields, com- petence, etc. Genetics, following a quite different mode of analysis, formu- lates its answer to the same question in a quite different way. It fmds that the development of the organ is dependent on the activities of certain genes in the fertilised egg. The task of this chapter is to present the picture of the development of an organ or tissue as seen in terms of genes. This will provide a view of the epigenetic system which we must take as being complementary to that derived from experimental embryology. I. Developmental pathways and their genetic control In using the data of genetics to throw light on the general character of developmental processes we are not concerned with the way in which any particular gene obtains its effect — a question we shall take up in the next chapter. Here we want to start from the other end, taking an organ or tissue and seeing how genetics would lead us to envisage its develop- ment. It will be convenient before discussing particular cases to summarise the general principles which we shall in fact fmd to emerge. The most important of these are : (i) The development of an organ or complex substance takes place in a series of steps, each of which is affected by genes. (2) At each step there are several genes acting, and the actual develop- ment which occurs is the resultant of a balance between the opposing gene-instigated tendencies. (3) At certain stages in the development of an organ, the system is in a more than usually unstable condition, and slight disturbances at such times may produce large effects on the later events. Such times have been called 'epigenetic crises'. (4) An organ or tissue is formed by a sequence of changes which can be called the 'epigenetic path' leading to it. In a normal egg which con- tains the genes usually found in the wild individuals of the species, these paths are rather defmitely distinct from one another, so that a developing 329 ^.Q PRINCIPLES OF EMBRYOLOGY mass of tissue turns either into a leg or into a wing say, but it is difficult to persuade it to become something intermediate And also each path i 'canalised', or protected by threshold reactions, so that if the developmen IS mildly disturbed it nevertheless tends to regulate back to the normal end-result (Waddington 1940^). As a first example in which these prmciples may be seen, we may con- sider the development of the wing in Drosophila (Goldschmidt, 193 5^, 1937, Waddington 1940^). The main features are shown m Fig. 15.1 m the form of somewhat diagrammatised drawings of the whole wmg at various stages during its development in the pupa. In the larva at the time of puparium formation, the wing is a thickened area of the dorsal meso- thoracic buds, an area which is already folded in towards the mterior of he bud Very shortly afterwards, the fold elongates and breaks through the thin* opposite side of the bud (stage 2) . The thick but pointed blade expands in area and becomes thimier, and as it does so, it becomes apparent that there are chamiels left open between the upper and lower surfaces; these are the pre-pupal veins (stage 3). Soon the wing, as well as expandmg in area, be- comes fatter by an inflation which forces the two surfaces apart, obhteratine^ the venation until the wing is transformed into a featureless sac (stage ^,. The greatest sweUing is reached just about the time of true pupation. From then onwards, the wing starts to contract again. As the two surfaces come together, they leave spaces between them: these are the adult vems which appear first near the tip of the wing, and gradually spread back towards its root (stages 5, 6, 7). At first the tissue between the vems is spongy and loose in texture, but gradually it becomes more compact the fluid which had filled the inflated wing sac being finally driven out to give an immature wing ahnost identical in outline with the final adult one, though smaller than it in size. In fact, after the last pupal stage drawn in the figure nothing much happens to change the morpho ogy of the wing except the expansion of the cells, throwing the whole structure Figure 15.1 The centre colunm shows eight stages in the development o£ th.Drosophda W during the pupal period, from the condition of an miagmal bud at the Top'throTgh theVriod of inflation to the aduk condmon^On the eft are thr e gene-controlled modifications affecting wmg shape: X.5f. produces a distallick as early as the imagmal bud stage; broad affects the direction of .rowth in the prepupal stage before the inflation ; Jmupy increases the longi- S contraction after the mflation. On the right are three genes altermg wing venation. Cubitus interruptus removes one of the P-P^P^l y-^-^^^^ causi the appearance of extra venation, and vemlet the obliteration of the dps of the normal veins, during the contraction followmg the mflation. (Adult wings to smaUer scale; after Waddmgton mob.) THE ROLE OF GENES IN THE EPIGENETIC SYSTEM 33I 332 PRINCIPLES OF EMBRYOLOGY into folds which only become flattened out again after the imago emerges from the puparium. The final wing is a simple-enough organ. It is practically two dimen- sional, since the thin upper and lower surfaces fuse tightly together; its outline is a simple oval slightly indented where the most posterior vein cuts it; and the whole system of venation consists only of one vein forming the fore-edge and four main longitudinal ones radiating from the base of the wing with two short cross-veins between them. The wing path of development is affected by very many genes ; Wad- dington (1940&) has described the abnormalities produced by some thirty of them, and a fair number of others are known. The mutant alleles of these genes are recessive to the wild-type alleles ; that is to say, the epigenetic path is canalised to the extent that an alteration of only one of the two alleles to the mutant form does not suffice to produce any notice- able alteration in the course of development, presumably because some threshold is not exceeded in the heterozygote. A few of the recessive forms are illustrated in Fig. 1 5 . i , which shows how their development diverges from the normal pathway. Each step in the normal sequence is influenced by genes, which often act upon the developing tissues in opposite directions. For instance, in the prepupal wing, the relative rates of cell division in different directions are affected by the genes broad, expanded, lanceolate and narrow, of which the first two cause the wing to become broader and the last two longer. Again, the time of the pupal contraction is a minor epigenetic crisis, during which the contracting wing is in a state of dehcate balance, influenced by genes such as dumpy, humpy and spade, which tend to increase the contraction in length, hlade which tends to increase it in width, balloon and bloated yAi\c\i tend to reduce the contraction in general; while genes whose primary effects are to change the shape of the wing margin may produce secondary effects at this time, since if the wing is abnormally long and narrow, or short and broad, before the contraction starts, these characteristics will become exaggerated. During the later stages of the contraction, the imagitial veins appear as cavities remaining between the two wing epith- elia and there are some genes, such as veinlet, tilt, radius incompletus, which tend to cause obliteration of veins, while others, such as plexus or net, work in the opposite sense, and produce extra veins. From a study of this large number of genes affecting the development of a single organ, a picture of the general epigenetic situation emerges. Direct investigation by more conventional experimental techniques has confirmed it in many points. Thus Lees (1941) made defects in the develop- ing wing by pricking it with a needle at various defmite times, and could THE ROLE OF GENES IN THE EPIGENETIC SYSTEM 333 interpret his results in terms of the same sequence of developmental mechanisms as the genetic study had revealed. Goldschmidt (1935^2) Henke (1947), Schatz (195 1) and others have treated Drosophila pupae v^ith just sub-lethal temperatures at definite times, and found that a series of abnormalities of wing development occur which parallel in a striking manner the forms produced by mutant genes. Such abnormalities which are similar to the phenotypes characterising genetic races but which are produced by environmental stimuh, are known as 'phenocopies . Henke found the interesting fact that the time at which a temperature shock was capable of causing the appearance of a phenocopy of some particular mutant type was usually near to, or just before, the time at which the development of that type first diverges visible from the normal. For instance, shocks just before or during the contraction phase tend to pro- duce dumpy- or hlade-\iik.Q. phenocopies. For each type of phenocopy there is a 'sensitive period' during which it can be relatively easily induced^ (Fig. 15.2). The fact that the sensitive period for the phenocopy of a gene occurs very near the time at which the development of the mutant becomes recognisably abnormal suggests at first sight that tliis is the stage of development at which the gene becomes active. It is, however, by no means clear just what might be meant by 'the time of action of a gene'. If an embryo contains a certain mutant gene, that gene is present in its cells from the time of fertilisation onwards. Long before there is any visible abnormality of development, the gene may have been producing some unusual substance which is merely stored up within the cell without being detectable by existing methods. The fact that an external agent can produce a given phenocopy most easily at a certain stage of develop- ment is a sign that the relevant epigenetic process is most unstable at that time. It is not surprising that it should be just at this time that the mutant gene also begins to produce detectable divergences from the normal course of development, but this does not tell us whether the gene has been active earlier or not. Nor, of course, are we justified in concluding that the gene acts by a mechanism similar to that of the environmental stimu- lus; in fact during a sensitive period many different external stimuli (heat, cold, x-rays, etc.) may produce similar effects, and it is clearly impossible that all of them can be disturbing the system in the same way that the gene does. Phenocopy studies can thus provide some information about the stability of the epigenetic situation in the developing tissues, and can ^ Occasionally there are two different sensitive periods, for instance when very similar end-results can be brought about in two different ways. 334 PRINCIPLES OF EMBRYOLOGY reveal the 'poiiit of attack' of a gene, but can as a rule tell us little about the gene's mode of operation or the time of its primary activity. The sensitivity of a stock of animals to environmental stimuli is under genetic control. One can, by selection over a number of generations, Figure 15.2 The curves above show the frequencies per cent of various types of pheno- copies produced when a temperature shock (3-5 hrs. at 39° C.) was given at certain times after puparium-formation to a stock of D. inelanogaster. The type of wings corresponding to the various curves are shown below, to- gether with the wild type ( + ). They are : (a) first broadened type ; {h) dumpy type; (c) first narrowed type; (d) rounded end type; [e) second broadened type, usually curved as well ; (/) normal proportioned but small ; (g) second narrowed type. (From Schatz 195 1.) THE ROLE OF GENES IN THE EPIGENETIC SYSTEM 335 build up Strains whose hereditary constitution predisposes them to respond in particular ways to any given treatments. This specification of the developmental stability of the organism is surprisingly precise and detailed; it usually depends, in any given case, on rather a large number of genes; and starting from any fairly large population, it seems to be always possible to find in it genes which will confer on their bearer almost any type of developmental reactivity one chooses. These facts again bring to our attention very forcibly the complexity of the genetic system which controls developmental processes. (For discussion of the evolutionary implications of phenocopy-formation and similar pheno- mena, see Waddington 1953 &, 1954&.) We may now turn to another example of a developmental sequence which has been well analysed from a genetical point of view (Lees and Waddington 1942, Lees and Picken 1945). The formation of the bristles (macrochaetae) in Drosophila appears, by histological investigation, to be an extremely simple process, directly involving only two cells per bristle^ (Fig. 15.3). In the pupae of about fifteen hours' age slightly enlarged cells may be found in the hypodermis. Aheady they are sometimes in pairs, though they may also occur singly. By nineteen hours they are always paired, and it is probable that a division, producing a pair of cells, occurs in the period from about twelve to eighteen hours. The two cells proceed to grow rapidly, attaining a volume about a thousand times as great as that of the neighbouring hypodermal cells. During this process the nuclei enlarge greatly, and the chromosomes assume the polytenic banded form which is best developed in the salivary gland cells. The pair of cells be- come arranged in a characteristic way, with one lying above and slightly to one side of the other. The upper cell, which is known as the tormogen, is destined to form a more or less circular socket of hardened chitinous material, while the lower one, the trichogen, produces a long gradually tapering bristle which sticks up through the centre of the socket. A whole series of genes affects this relatively simple sequence of processes. In the first place there are some, such as scute, which cause an absence of certain of the normal bristle cells, and others, such as hairy, which produce extra pairs; nothing further is knowoi about their mode of action. The next stage, that of the cell division to produce the trichogen-tormogen pair, is affected by split, which often provokes an extra division to give a group of four cells; these become somewhat irregularly arranged, and may give rise to two trichogens and two tormogens or only one of the ^ There are one or more other pairs of cells closely associated with these ; a general description is given by Henke (1953) • 336 PRINCIPLES OF EMBRYOLOGY N M Figure 15.3 The genetic control of bristle development in Drosophila. A and B are sec- tions through the thoracic hypodermis of the mid-pupa, showing two stages in the development of the bristle-forming (trichogen, trch) and socket- forming (tormogen, tor) cells. C, surface view. D, surface view showing the extra division which occurs in split, giving (£) duplicated bristles. F, surface view, and G, section, in Hairless, when the trichogen and tormogen lie side by side at the same level, and give two sockets (H). In shauen-nakcd {I, J,) the situation is similar but usually not so extreme. In Stubble {K, L, M) the tormogen does not embrace the trichogen as closely as usual, and an ab- normal thick bristle is produced. N shows the irregular bristles produced by ^orked. (From Lees and Waddington 1942.) former and three of the latter. Another gene, dichaete, has a somewhat similar action, but it is usually only the tormogen which divides a second time and becomes doubled, hi the next step, the precise arrangement of the tormogen above and to one side of the trichogen is also under genetic THE ROLE OF GENES IN THE EPIGENETIC SYSTEM 337 control. In hairless, shaven-naked and prickly, both cells lie side by side at the surface and both produce sockets, so that no hair is formed; while in stubhle the tormogen is shifted slightly to one side and sits less firmly on the trichogen, and the hair is thicker and shorter than normal. These phenomena indicate that the shape of the bristle is at least partly due to the moulding of the still-fluid secretion of the trichogen as it is forced through the constricting ring of the tormogen. Several genes affect the nature of this secretion and the rate of its production. Spineless and morula reduce its amount considerably, so that only short and thin bristles are formed, and the secretion of the trichogen is also reduced to the level characteristic of the tormogen in those mutants in which both cells lie side by side at the surface {hairless, shaven-naked, prickly). In stubble the early rate of production is increased but the fmal value is much the same as in normal animals. Some other genes have a more subtle effect. During its secretion the bristle seems to consist of a thin plastic wall and a more fluid core, the whole of which eventually hardens and becomes hollow. The wall is probably formed of an oriented long-chain high-polymer in a rubber-like state, almost devoid of cross-linkages between the chains. It normally grows just at the rate required to provide enough surface to accommodate the fluid secretion which is being forced into it by the trichogen. Tliis delicate balance between growth in surface and in volume is disturbed by certain mutant genes. In singed and forked the volume may suddenly increase too fast (or the surface too slowly) so that the bristle 'explodes' and forms bulges and kinks, while in bristle there are gradual and periodic changes in the volume surface/relationship, so that a series of fairly gentle swellings arises. Moreover these genes also effect the mech- anical properties of the surface, which is more plastic in singed sLnd forked and more elastic in bristle. Many of these mutant types of bristle may also be produced in pheno- copies, but the correlation between the sensitive periods and the points of attack of the genes is not so fidly worked out as for the wings. An even more impressive demonstration of the real complexity of apparently simple developmental processes is provided by the chemical investigations which have been made in recent years on the eye pigments o£ Drosophila (Beadle 1945, Ephrussi 1942, Nolte 1952) and various other insects, e.g. the meal worm Ephestia (Kiihn 1941, Caspari 1949) and the silkworm Bombyx (Kikkawa 1953). In Drosophila at least twenty-five genes are known to affect the colour of the eyes, which normally are a slightly brownish red. Analysis of the effects produced when two different mutant genes are both present in homozygous condition led to the suggestion that there are two main pigments, a brown and a red, 338 PRINCIPLES OF EMBRYOLOGY and these were later found to be separately extractable with suitable sol- vents. They are both of relatively low molecular weight, though probably bound to proteins when they are in the natural state in the living cell. Rather little is known about the development of the red pigment, except that it fails altogether in the absence of the normal allele of brown {bw+) and is affected less profoundly by many different genes. Chromato- graphic analysis has quite recently shown that it is in fact really a mixture of a rather large number (about ten) of different components (Heymann, Chan and Clancy 1950) and that certain other fluorescent compon- ents are closely associated with it (Hadorn 195 ifl, Hadom and Mitchell 1951). The production of the brown pigment provides a good example of a sequence of developmental steps, and is particularly interesting because it has been possible to discover the actual chemical changes involved in some of these. Sturtevant (1932) pointed out that in a fly heterozygous for vermihon (t^/j^+) it can sometimes happen that the v+ chromosome gets lost at a mitosis, and that a patch of cells with the constitution v may appear among the heterozygous eye facets, which are normal in colour. When this happens with most other genes, an abnormally coloured group of cells can be clearly seen, but no departure from the normal wild-type pigmentation is found in patches which can be shown, on other evidence, to be V in constitution. Sturtevant therefore suggested that some substance diffuses into these cells from the surrounding tissue and compensates for the absence of the y+ gene. Beadle and Ephrussi developed a technique of transplanting pupal eye- discs into the body-cavity of other larvae, and by suitably choosing the genotype of the host and of the transplant, were able to show that there are at least two diffusable substances (which they rather unhappily called 'hormones') concerned in the production of the brown pigment. One of these is produced under the influence of the normal vermillion gene (f +) and is lacking if that gene is replaced by the mutant-vermilhon allele (v), while the other is similarly related to the cinnabar gene. Investigation finally showed that these substances are derived from tryptophane, which is converted first (under the action of v+) into a-oxytryptophane, which is then oxidised (by a reaction for which no separate genetic control has been identified) to kynurenine, which was before its identification known as the v+ substance. This in its turn is converted, under the influence of the cn+ gene into a 'm+ substance , which is probably 3-hydroxy kynurenine; and after this two further steps of reaction, controlled by the normal- cardinal and normal-scarlet genes, intervene before the actual brown pig- ment is formed (Fig. 15.4). THE ROLE OF GENES IN THE EPIGENETIC SYSTEM 339 The reactions leading to the brown pigment must be hnked in some way with those leading to the red, since some genes, particularly those of the white locus, affect them both. Probably this gene is essential in connection with some common substrate or carrier-protein, which has to link up with the m+ substance and with something equivalent in the reaction-series leading to the red pigment. It is interesting to note that the cn+ substance can only produce its effect during a certain limited period of development, extending from sometime after puparium formation for about forty- eight hours. Presumably before that time the kynurenine is not yet avail- BROWN PIGMENT f,cd+, CHROMOGEN (cn* subsl-once) RED PIGMENT bw^QENE-- PRECURSOR a. C-CHiCHCOOH NKt J^ (y* subsVanco.) Cx?- CHfCH-COOH NHt 'COMMON: SUBSTRATE, CARRIER, OP ENJYME *— W^OBNE CC-OXVTRYPTOPHAN TRYPTOPHAN Figure 15.4 Eye pigment development in Drosophiln. (After Beadle 1945.) able, while by the end of the period of sensitivity it must have been con- verted into something else. This provides a clear parallel to the restriction of periods of competence found in more usual experimental embryological studies. It is also worth pointing out that phenocopies of eye-colour mutations cannot be easily produced by environmental shocks of a general nature, such as high or low temperatures; the epigenetic system is not complex enough to be unstable with respect to such non-specific agents. Another well-analysed system of pigmentation is that of the guinea- pig hair colour (Wright 1942). It will not be described here but it well illustrates the point which is being made, namely the real complexity of apparently simple developmental processes. 340 PRINCIPLES OF EMBRYOLOGY There is a further important point about developmental paths, or se- quences of epigenetic reactions, which has not been adequately illustrated by the examples so far described. That is the existence of sharp distinc- tions between the comparatively small number of alternative paths which normally occur in the development of a particular species. In embryological studies, we have seen (p. 179) how amphibian gastrula ectoderm is competent to become either epidermis or neural tissue or mesoderm, and in the great majority of cases becomes quite defmitely either one or other of them. A similar situation is revealed by some genes in Drosophila. For instance, Goldschmidt (1938, 1945) has shown that in flies of certain abnormal genotypes, what would normally be the pre- sumptive wing tissue enters on one of the alternative epigenetic paths, and develops as a haltere; or in another genotype it becomes a leg. If the normal aristopedia gene is replaced by its mutant allele {ss"), the tuft on the antenna known as the arista may develop into the terminal section of a leg, with perfectly developed claws. Genes which produce this type of effect, in which a part of the embryo develops into an organ which is normally located somewhere else in the body, are known as homeotic genes. A considerable number of them are known in Drosophila. Besides aristopedia, for instance, prohoscipedia con- verts the mouth parts into a leg-like organ; tetraltera converts the wings into halteres, while tetraptera converts the halteres into wings; podoptera causes the wings to become legs, while hithorax changes the whole meta- thorax into a mesothorax. Some of these effects can also be produced by environmental agencies; thus Gloor (1947) found that ether treatment of the young larva a few hours after laying would produce a phenocopy of hithorax, presumably by changing the condition of the larval hypodermis at the time when the imaginal buds are first being formed. Similar changes can, however, also be produced at a much later stage since, as we saw (p. 141), abnormalities in the folding of the imaginal buds just before pupation may cause parts of them to develop into tissues which should belong to another part of the body. It is probable then that most of the homeotic genes act in the first place at the time imaginal buds are first formed but that these alterations do not produce any defmitive effect until about the time of pupation. Once a piece of tissue has entered on a path of development, its final condition can be affected by all the genes which are concerned in that path. Thus, if, in an animal in wliich the arista takes the leg path owing to the presence of the mutant 55" gene, the genes concerned with the develop- ment of the leg have been replaced by mutants such as fourjointed wliich produce shortened legs, then tlie aristal leg will also be shortened; while THE ROLE OF G'ENES IN THE EPIGENETIC SYSTEM 341 genes which usually affect the arista will now have no effect on it (Wad- dington 1940c). This shows that, as might be expected, the activity of a gene is not determined simply by the geographical location within the body, but by the type of developmental process which is going on (Fig- 15-5). ^% m:^ ss' th S5 Figure 15.5 The mutant allele ss"^ in Drosophila converts the whole arista into a leg, while the allele ss"^ changes only the proximal part into a leg, the distal part remaining an arista. A gene th (thread) which normally removes the branches from the arista, affects only the arista-like portion in ss"^ flies, andj^' (four-jointed) which shortens the legs also shortens the leg-like arista of 55'''\ (From Waddington 1940a.) Primary and secondary effects of genes Some of the studies we have mentioned, particularly that of Beadle and Ephrussi on the eye colour genes, were at one time thought to hold out the hope of leading us to an understanding of the primary, immediate effect of a single gene. This has turned out, so far, to be illusory. It has in no case proved possible to be absolutely sure that the gene-effect we can see is the primary one. The subject is discussed more fully later (p. 379), but it is important at this stage to consider some general points about the kinds of primary and secondary effects which genes may have on the development of organs and tissues. During the development of a complex animal, any alteration pro- duced by a gene at an early stage may have many later repercussions, perhaps in quite other organs than that in which the original effect occurred, A very obvious case is that in which a gene affects an organ of internal secretion. For instance in the mouse a certain gene impairs the secretion of the growth hormone by the pituitary, and, as might 342 PRINCIPLES OF EMBRYOLOGY be expected, this affects all these structures for which the hormone is important, so that the animal develops as a dwarf. Griineberg (1948) has shown that many other cases in which a gene has manifold and widespread effects can be explained in a similar way. One of the best-known examples is that of a certain lethal in rats. Animals homozygous for the gene show a very diversified complex of symptoms, which eventually lead to their death at an early age. Griineberg (1938) argues that all (or nearly all) the symptoms can be plausibly considered to be secondary consequences of an original hypertrophy of the cartilage. The network of causation which he postulates in this case is shown in Fig. 15.6. Falconer, Fraser and King (195 1) have described another case, in which a gene 'crinkled' in mice produces a large number of different effects which they suggest can be almost entirely accounted for as the results of a suppression of hair follicle formation between twelve and a half and seventeen days of gestation and after birth. The suggestion that all the effects in this case are actually secondary consequences of one initial abnormality receives very strong support from the fact that another different gene tabby also produces (in single dose) exactly the same syndrome. If each symptom was brought about by a separate reaction of the gene, such a parallelism could not be expected unless the crinkled and tabby genes were identical, which the genetical evidence shows them not to be; whereas if there is one single underlying cause for the syndrome, it is easy to imagine that two or more different genes may affect the basic process and thus produce the same complex end-result. Hadom (1948^7, 1950, 1951^) has paid particular attention to the 'pattern of manifestation' of a gene, i.e. the particular collection of organs whose development it alters. He studied certain 'lethals' in Drosophila, that is genes whose effects are so profound that individuals homozygous for them do not survive. He emphasises, first, that there are critical periods of development at which death tends to occur. Thus many lethals are known which produce death at the end of the embryonic period, and again there are many for which the time of death is at puparium forma- tion, or at emergence; but there are very few which kill during the middle of larval life. These sensitive periods are the times when a great deal is going on in the epigenetic system, so that slight abnormalities in the tissues, which may have been produced considerably earlier, will then cause drastic effects. They have also been referred to as 'epigenetic crises', and are of various grades of severity. Hadom went on to show that if the phenotypes produced by the lethal genes are closely examined, each gene wiU be found to cause a characteristic pattern of damage, exhibited either by the death and THE ROLE OF GENES IN THE EPIGENETIC SYSTEM 343 "Anomaly of cartilage" Narrowed lumen of trachea Thickened ribs Fixation of thorax in inspiration Slight changes in larynx and nose Kyphosis Abnormal situs of thoracic viscera Spur on deltoid ridge of humerus Slow suffocation Increased resistance General arrest Blocked nostrils in pulmonary of development circulation Coma exposure inanition Compensatory hypertrophy of right ventricle of heart Blunt snout Inability to suckle Decompen- sation Exitus Capillary Faulty occlusion haemorrhages of incisors into lungs Difficulties in feeding Starvation V Gxitus Y Exitus Exitus Exitus Figure 15.6 A gene in the rat causes abnormalities in the formation of cartilage. This has many secondary consequences ('pleiotropic effects'). (After Gruene- berg 1938.) 344 PRINCIPLES OF EMBRYOLOGY degeneration of the cells of particular tissues, or, when the effect is weaker, by a retardation of growth. This pattern differs in detail in different stocks of the lethal, depending on the other genes associated with it (the 'genetic background'). More important from the present point of view is the fact that the pattern in which the gene normally manifests itself is a mixture of primary and secondary effects. That is to say, in some tissues the abnormality is a direct consequence of the activity of the lethal gene in those particular cells (a primary effect), while in other tissues the gene may either be inactive, or ineffective because its action fails to surpass some threshold, yet the tissue may develop abnormally because it is influenced by unusual substances produced elsewhere in the body (a secondary effect). Hadom demonstrated the reahty of such secondary effects by showing that certain organs from larvae of a lethal type will continue to develop more or less normally if transplanted into host larvae of a wild-type strain. For instance, lethal-meander and lethal-translucida both die usually at about the time of puparium formation; but the imaginal buds, if transplanted into normal hosts, can carry through a complete metamorphosis. Their death when left undisturbed is therefore a second- ary consequence of abnormalities in the development of other parts of the body (probably the protein-metabolising system in the gut of lethal- meander and some other nutritive or hormonal peculiarity in lethal- translucida). The distinction between primary and secondary effects can of course also be made within the confmes of a single organ. For instance, if the primary effect of a gene is to cause the absence of large parts of the anterior and posterior regions of the wing (e.g. Beadcx or Lyra) a second- ary consequence is that when the pupal contraction occurs the longi- tudinal veins become squeezed nearer together and diverge at a smaller angle (Waddington 19406).) The fact that in Hadom's cases the secondary effects occur in different organs to the primary ones is not the essential point of the distinction, but merely makes it easier to recognise which effect is nearer the very first influence of the gene on the sequence of developmental processes. Hadom has provided a diagram, reproduced in Fig. 15.7, which ex- presses neatly some of the ways in which secondary effects may occur. In this, the rectangles i, 11 and m represent three cells in three different organs, as it might be Imaginal Bud, Fat Body and an Endocrine Gland. In each cell ten genes are represented. It is supposed that in each type of cell the heavily ringed genes are active, producing substances (large rings) which fit together into a reaction-chain. Consider what would happen if various of these genes mutated. If the activity of 8 was altered, nothing THE ROLE OF GENES IN THE EPIGENETIC SYSTEM 345 would be changed except in organ i, where a primary manifestation would occur. If 3 mutated, that would cause a primary effect in iii and a second- ary one in I, in the reaction-chain of which an essential link is a product of III, the 'hormone' b. If, before the lack of b had had irreversible conse- quences, organ i were transplanted into another host with unmutated 3 , then supplies of b would be available to it and it would develop normally. Again, mutations in 4, 5 and 6 would cause secondary effects in i and m as 1 11 I Figure 15.7 Diagram of a sequence of gene reactions in three different tissues. See text. (From Hadorn 1950.) well as a primary one in 11. But if a mutation occurred in genes i or 2 or 10, both organs i and m would be altered, and these alterations would both be primary ones. When a gene affects several different organs or tissues it is said to be 'pleiotropic'. The term is not always a very precise one, since in border- line cases there may be difficulty in deciding whether two effects are to be considered the same or different (e.g. in Beadex or Lyra above). The con- cept has, however, been widely discussed, since it clearly is of great im- portance to discover whether any one gene can only exert one type of 346 PRINCIPLES OF EMBRYOLOGY immediate effect, or whether a single gene can do different things. It is clear there is no evidence for more than one type of gene-action in cases where the whole of a complex pattern of manifestation can be shown to consist of a set of secondary consequences of some single primary effect (as in the mutation of 3 above, or Griineberg's rat lethal described on p. 342). Griineberg speaks of this as 'spurious pleiotropy', contrasting it with 'genuine pleiotropy', in which the same gene would be doing different things in different tissues. The difficulty with this latter category, however, is that at present we have no certain way of detecting it. We can discover cases which seem to be of the type produced by a mutation of gene i, i.e. in which there are two or more apparently primary effects which are not influenced by other tissues. But there is no way of telhng whether the mutated gene i is doing the same thing in organ i as it is in organ iii, and we can at best class this as an 'apparently genuine pleiotropy'. Most attention, from the embryological side, has recently been given to cases of spurious pleiotropy, since the secondary effects may reveal some of the epigenetic interrelations between tissues. The primary effects, however, are also not without interest. In a case of 'apparently genuine pleiotropy' we are confronted with two or more at first sight disconnected developmental processes, which are shown actually to be related by the fact that one particular gene is involved in all of them. They must therefore have some fundamental similarities ; and the nature of these offers a very important problem which may go to the very heart of the epigenetic systems involved. To take an example. In Drosophila, split causes both an extra division in the bristle-forming cells (followed by various abnormalities in their arrangement), and also an effect on the facet-forming cells of the eye which is in the main a deficiency in the normal number of cell divisions (accompanied also by some irregularity in arrangement). Now the facet-effect o£ split is almost exactly mimicked by morula, another effect of which is to reduce the growth rate of the bristle-producing cells so that the chaetae are smaller than normal. This bristle-effect of morula, in turn, is mimicked by spine- less; and many of the alleles at the spineless locus cause as well the 'aristo- pedia' phenotype, characterised by the conversion of the arista into a leg, the basic alteration being perhaps one on the growth rate and folding of the imaginal bud, We have then a series of developmental reactions, revealed by the mutant phenotypes of split, morula, and aristopedia, which would seem quite disconnected from one another were it not that the overlapping pleiotropies show that there are some basic relationships at the level of apparently prhnary gene action (Lees and Waddington 1942, Waddington and Pilkington 1943). THE ROLE OF GENES IN THE EPIGENETIC SYSTEM 347 Genes may also be used iii another way to reveal basic relationships between developmental processes, namely by breeding animals which simultaneously show the effects of the two genes which one wishes to compare. For example, it has been mentioned that in hairless and shaven- naked some of the trichogens and tormogens are shifted so as to lie side by side, while in stubble there is sHghter effect in the same sense in all the cell- groups. Now in hairless-shaven flies the effect is very strongly exaggerated, and occurs in nearly every group, while in stubble-hairless or stubble-shaven there is a straightforward summation of the two effects. From this one may conclude that the actions of hairless and shaven belong to one group and those o{ stubble to another. One camiot be certain of the nature of the relationships within and between groups, but it has been suggested that genes which show exaggeration when combined are those which act at the same time on the same epigenetic process ('homodynamic' genes), while with those which act at different times the buffering of the system reduces the severity of the effects (Waddington 1953). SUGGESTED READING Beadle 1945, Ephrussi 1942, Goldschmidt 1938, pp. 3-98, Hadorn 1948a, 1950, Griineberg 1948, Waddington 1940b, 194.8a, Weiss 1947, i9Sob, Wright 1941 or 1942. CHAPTER XVI THE ACTIVATION OF GENES BY THE CYTOPLASM IT HAS frequently been argued that genes control only the later-developed and more superficial characters of animals and that the development of the basic plan of the body is controlled, not by them, but by the cyto- plasm of the egg; and this contention has been hotly disputed by genetic- ists w^ho seem to feel that it disparages the importance of their subject. We realise now that, as in so many such controversies, both sides are in the right. Undoubtedly within any one lifetime a great deal of the basic pattern of the body is dependent on the configuration of the cytoplasm of the egg; one need only remember such phenomena as the arrangement of ooplasms in mosaic eggs (p. io6), of gradients in the echinoderms (p. 85), or of the formation centre in insects (p. 126). There are indeed many cases in which genetic differences in the nuclei can be shown to have an influence even in early stages of development (e.g. in the merogons or hybrids in frogs, p. 358), but they are certainly not all-important. Thus for embryology the cytoplasm is as fundamental as the genes. Equally, of course, one must not forget that an egg will not develop into even the general framework of an animal unless it is provided with nuclei. There can be no doubt that differentiation results from the inter- action of the division-products of the original zygotic nucleus with the already-present ooplasmic regions of the egg. Neither cytoplasm nor nucleus can be disregarded: in fact the most important subject to discuss is how they affect each other. We can, then, put into the following form the knowledge with which we have to approach the problem of how development is brought about: (i) There are local differences with the cytoplasm of the newly fertil- ised egg. The nuclei, with the genes contained in them, react with the cytoplasm with which they are in contact; and the interacting system of nucleus and cytoplasm may also be affected by substances diffusing from neighbouring regions, as in the process of induction. (2) As a result of the nucleus-cytoplasm reactions there are formed, firstly new duplicate genes (which allow for the multiplication of nuclei), and also substances of some kind which pass into the cytoplasm and modify it. (3) These 'immediate gene products' may interact with each other in 348 THE ACTIVATION OF GENES BY THE CYTOPLASM 349 the cytoplasm, or with other substances which are already there. There is likely to be quite a complicated series of reactions of this sort before the appearance of the differentiated cytoplasmic constituents which charac- terise the various fully developed tissues of the adult animal. As these reactions go on, the cytoplasmic environment of the nucleus will be altered, and this wdl affect the nature of the nucleus-cytoplasmic re- actions. The gradually changing constitution of the cytoplasm may also be expected to alter the nature or intensity of the activities of the im- mediate gene products. We must therefore be prepared to fmd ourselves faced with a double cyclic system, of the kind pictured in Fig. 16.1. IMMEDIATE GENE PRODUCTS CYTOPLASMIC PROTEINS Figure 16.1 The double cycle of intra-cellular reactions. The genes in the nucleus acting on cytoplasmic substrates, both reproduce themselves and control the form- ation of 'immediate gene products', a, b, c, etc. These then use cytoplasmic raw materials (i) perhaps to reproduce themselves identically, in the mamier of plasmagenes, and (ii) to elaborate the final cytoplasmic constituents, P, Q, R etc., which are the substrates or raw materials which condition the activity of both the genes and the gene products. (From Waddington I954fl.) (4) The whole system is organised in such a way that development tends to proceed along one or other of a number of alternative canalised paths. That is to say, the developmental reactions tend to go towards one defmite type of end-result or another; and while development is going on, the system has some power of self-regulation, so that no efiect is 350 PRINCIPLES OF EMBRYOLOGY produced by minor abnormalities such as slight losses of material or the substitution of one recessive gene for a dominant one. It is now necessary to discuss in rather more detail the successive phases in this system of reactions. We shall leave till a later chapter the questions relating to morphogenesis in the strict sense, that is to say, the mould- ing of the developing tissues into defmite shapes. Here we shall be con- cerned with the processes of chemical change during differentiation. This is one of the most active, important and controversial fields of biology at the present time. There are many different theories which wiU need to be considered; and since the problems get so far down to the common root of aU biological phenomena, light may be thrown on them from very many different angles. We shall have to consider factual material drawn not only from experimental embryology of the old-fashioned kind, but from biocheinical studies and from the genetics of micro- organisms as well as higher forms. If is often difficult to assess the relative importance of these various types of evidence. On the one side we may have what seems very precise and defmite biochemical information, expressed in terms of nucleic acids, enzymes, phosphate bonds and so on — but we have to ask ourselves just what is the connection between this and the phenomena of development which we are trying to explain, and con- sider whether it really is more enlightening than theories which operate with less clear-cut concepts (ranging all the way from genes to organisers and ooplasms) which are further from cheinistry but nearer to the em- bryos. The fourth point mentioned above may be dealt with first. It is a platitude, but an important one, that the body of a multicellular animal is made up of tissues which are rather distinct from one another. Even when changes in the normal course of tissue differentiation are brought about (e.g. by induction, or the action of lithium on the amphibian mesoderm, etc.) the altered tissue is usually switched from one into another of the well recognisable types — from epidermis to neural tissue, or chorda to somite. Intermediate types occur rarely, and when they do (as for instance the 'palisade tissue' found as a 'weak' reaction to a neural-inducing stimulus) they often later develop into something more normal, such as ganglion tissue in the case of palisades. The developmental reactions, therefore, tend to follow one or another of a number of definite paths, which lead to rather well defined and distinct end-states. Further, there is abundant evidence from all the regulatory phenomena which are so common during development, that even if the conditions in a developing tissue are made somewhat abnormal, the epigenetic system is often able to compensate for this, so that the normal end-state is nevertheless THE ACTIVATION OF GENES BY THE CYTOPLASM 35I attained. Both these points can be expressed visually by means of a diagram such as that of Fig. 16.2, which represents what has been called the 'epigenetic landscape' (Waddington 1940a, q.v. for further discussion). I. The effects of cytoplasm on the nucleus In earlier chapters we have seen several instances in which localised areas of cytoplasm must have exerted an effect on the nuclei which move Thora« Wlitf Upp ^» ^2/3 I CHNH- I COOH CO2 ^ urease 2NH3 , proteins Figure 17.4 Metabolic pathways leading to the synthesis of arginine, with indications of the points where different genes in Neiirospora (i, 2, 3, 4, 5, 6, 7) interfere. citrulline or arginine) ; 5 and 6 can grow if provided with citrulline or arginine, but ornithine does not make good their deficiency, while 7 requires arginine and cannot make do with either of the other two substances. Very many similar situations have been discovered. Since many of the steps in these sequences are knov^m or believed to be carried out by means of enzymes, it is natural to assume that the funda- mental activity of the genes involved is the production of these enzymes, and that when the gene mutates the enzyme is either not produced at all or appears in an inactive form. In most cases this suggestion remains a hypothesis; it is rarely that the enzymes have actually been mvestigated, and when this has been done it has sometimes been found that the relevant one is not absent from a strain in which some reaction-sequence is inter- rupted, but is present though not effective (Wagner 1949). Nevertheless the postulated gene-enzyme relationship has been generalised and made 380 PRINCIPLES OF EMBRYOLOGY into the so-called 'one-gene-one-enzyme' theory. This supposes that all genes operate through the medium of enzymes, and that each gene is con- nected in the first place with one and only one enzyme, hi one form of the theory the gene is supposed actually to produce the whole enzyme; in another the suggestion is only that the gene determines the specificity of the enzyme, and thus its abihty to react, and its efficiency in reacting, with the appropriate substrates. This hypothesis has been widely beheved in recent years, and even when some doubts are expressed as to its adequacy, it has usually been given credit for having stimulated a great deal of valuable work. Actually, however, it is probable that the stimulus to the work came rather from the experimental technique of identifying mutations which lead to the block- age of a series of synthetic reactions rather than from the one-gene-one- enzyme hypothesis itself. In fact, almost the first problem which the hypothesis would raise is whether a given gene necessarily controls the formation of the same enzyme in different tissues, in which it is reacting with different cytoplasms. This question, however, still remains quite unanswered and hardly tackled, not, surely, because it is far removed from theory but because it demands a different experimental approach. The gravest criticism of the one-gene-one-enzyme theory is that it draws its support almost entirely from studies of unicellular or very primitive organisms and thus leaves out of account of the whole range of phenomena involved in regionahsation, which may or may not fall into hne with it. Even within the realm of the micro-organisms, it seems that at the present time the theory is beginning to appear as an over- simplification (cf Haldane 1954). However, work of the kind associated with it has led to the production of numerous genes which have thrown light on a wide variety of problems. For instance, it has been shown that a gene which has mutated to an apparently inactive form may many generations later mutate back again, and the corresponding activity reappears in the cells. This shows that a gene may continue to multiply in a sequence of dividing cells even though it shows no signs of activity; though of course it remains doubtful whether the gene is truly inactive or is continuing all the time to produce an enzyme of altered specificity. Again, this material provides some relatively clear-cut examples of secondary interactions between gene-controlled processes of the kind which seem necessary as a basis for a general theory of development (cf. Chapter XIX). Thus in Neurospora a certain gene blocks the formation of isoleucine and this leads to the accmnulation of its precursor, which cannot be utiUsed but increases in concentration until it inhibits a different but connected reaction by which a-ketoisovaleric acid is changed into THE SYNTHESIS OF NEW SUBSTANCES 381 valine (Bonner 1946). Another type of interaction is exhibited by the fact that in certain strains of Neurospora and Aspergillus which require an external source of L-arginine, an addition of L-lysine to the medium reduces the effect of the added arginine; and the same competitive in- hibition between the two substances is shown by a lysine-requiring strain (cf. Pontecorvo 1950, Emerson 1950). Finally, we may quote from another field a biochemical example of competition between gene-produced substances (or, less probably, the genes themselves) for a common substrate. One of the earliest attempts to link up gene action with known chemical compounds was the investiga- tion of the genetic control of flower colour (Lawrence and Price 1940, Haldane 1941, 1954). Genes were identified which caused various pre- cisely known chemical changes in the constitution of the coloured sub- stances. Probably these genes act by controlling the formation of enzymes; the evidence is no better, and not much worse, than it is for the similar hypothesis in the Neurospora work. Now in crosses involving a number of genes affecting the various different classes of pigments (anthocyanins, flavones and chalkones) it was found that there were interactions in which all the different types of substance were involved. When much antho- cyanin was formed, this led to a reduction in the amounts of flavone and/ or chalkone. Thus the different gene-activities competed, with efficiencies corresponding to the number and strength of the genes involved, for a limited amount of a common substrate from which the compounds of all those types were derived (Lawrence 1950). 5. The synthesis of proteins Since most, if not all, enzymes are proteins, the evidence from genetics that genes control the formation of enzymes should be linked with such general information as we have about the synthesis of proteins in cells. Although the nature of the chemical processes involved in this synthesis is still almost entirely obscure, there is quite a large amount of rather indirect evidence on the role which various parts of the cell structure play in the production of proteins. Most of this does not come from in- vestigations which have used embryos as the experimental material (for which see ten Gate 1953 and Gustafson 1952) but from studies in the general field of biochemistry and physiology. It is, in the first place, fairly generally agreed that the nucleic acids participate in an important manner in protein synthesis and indeed are probably essential for it. The synthesis of new chromosomal proteins, which is involved in the reduphcation of the genes before cell division 382 PRINCIPLES OF EMBRYOLOGY takes place, never occurs in the absence of desoxyribose nucleic acid. In the synthesis of cytoplasmic proteins, it is ribose nucleic acid which is thought to play the active part. Chemical analyses always demonstrate an unusually high concentration of RNA in rapidly growing cells or in those which are actively secreting protein (e.g. some glands, hair-forming cells, etc.). This RNA is largely located in the cytoplasm, but there is often also a considerable enlargement of the nucleoli, which are rich in RNA. For example, nucleoh are absent in the cleavage cells of the amphi- bian neurula, but appear at about the time of gastrulation, when there is evidence that specific proteins begin to be produced. There are at present two main theories about the processes of protein synthesis in embryonic or developing cells. The first is that of Caspersson (Reviews : 1947, 1950). It is based chiefly on studies which use spectroscopic methods to take advantage of the fact that the purine and pyramidine bases incorporated in the nucleic acid molecule have very characteristic absorptions in the ultra-violet; this makes it possible for the nucleic acids to be identified within living cells, although it must be pointed out that there are considerable technical difficulties and the method has come in for a good deal of criticism. From his observations, however, Caspersson has come to the following conclusions. The euchromatin, which consists largely of histone and DNA, synthesises replicas of itself and also produces other complex proteins, which could be the agents through which gene- action is exerted: however, the spectroscopic evidence does not suffice to suggest much about them. The heterochromatin (or, more generally, the 'nucleolus-associated chromatin') which contains an important pro- portion of RNA, is supposed to control the nucleic acid metabolism of the whole cell. It also produces proteins, which tend to be rich in di- amino-acids. These accumulate in the nucleolus, and diffuse from there to the nuclear membrane. On the outside of this, an intensive production of RNA-protein takes place and this is the main source of the cytoplasmic proteins, which are thus supposed to be formed in the immediate neigh- bourhood of the nucleus (Fig. 17.5). The other main theory is that of Brachet (cf. 1950, 1952). It differs from that of Caspersson in being derived from a large variety of biochemical, cytological and embryological investigations rather than from a single technical method such as spectroscopy. In its conclusions it lays much more stress on the role of the various types of cytoplasmic particle in protein synthesis. Brachet supposes indeed that the ribose nucleotides formed by the nucleolus (or heterochromatin) do not merely combine with proteins in the neighbourhood of the nuclear membrane, but take part in the formation of microsomes scattered throughout the whole THE SYNTHESIS OF NEW SUBSTANCES 383 cytoplasm, and that it is at these particles that the main synthesis of cytoplasmic proteins occurs. There is as yet, perhaps, no evidence which finally settles the question of whether cytoplasmic synthesis is directly under the control of the nucleus or whether the microsomes are essentially involved. The import- ance of the nucleus, either at first or second hand, can of course not be FiGunE 17.5 Protein synthesis according to Caspersson. A, the 'nucleolus-associated chromatin', containing ribo-desoxy-nucleotides (RDN), proteins (P), di- amino-rich proteins (DP) and perhaps ribose-nucleotides (RN). B, the nucleolus, containing DP and smaller amounts of RN. C, the nucleus, in which there is a gradient of P and DP towards the nuclear membrane D. In the cytoplasm E there is a gradient of P and RN from the nuclear mem- brane outwards. (After Caspersson 1950.) denied. For example, Weiss and Hiscoe (1945) have shown that in a neuron growth does not take place throughout the enormously elongated axon, but synthesis occurs only in the cell body in the neighbourhood of the nucleus; the new cytoplasm flows from this region towards the tip of the fibre, and if the axon is constricted, the flow becomes dammed up and a swelling appears proximal to the constriction (Fig. 17.6). The cell body is, however, also the region in which the cytoplasm is most baso- philic and probably the site of the main concentration of microsomes, so 384 PRINCIPLES OF EMBRYOLOGY that it remains quite possible that the influence of the nucleus is indirect and mediated through them. Again, Brachet and Chantreime have shown by radioactive tracer studies that if one removes the nucleus from the large unicellular alga Acetabularia (cf. p. 377), protein synthesis eventually de- creases in the non-nucleated fragment (cf. Brachet 1954). But it is remark- able that the activity continues unaltered for almost a fortnight and re- mains very considerable for much longer; from this they draw the ^■■■H Figure 17.6 Diagram of nerve regeneration. Rows ^ to jE show the normal process when the nerve is simply cut; regeneration takes place from the proximal portion. Rows F, G, H show that if, after the stage of row D, the fibre is constricted, the new cytoplasm being synthesised in the proximal (nuclear) end of the cell forms a swelling. When the constriction is removed (/) this cytoplasm gradually spreads distally. (After Weiss and Hiscoe 1948.) conclusion that the nuclear control of protein synthesis is indirect. The final proof that the microsomes take part in the process, and are the actual site of the new production of protein, would be given if they could be transplanted to some unusual location, say to another cell of different developmental fate, and it was found that the protein characteristic of the transplanted material was produced in the new position. Attempts to do this with isolated microsomes have so far been unsuccessful. The results of high-speed centrifugation of ascidian eggs (p. J14) or of echinoderm eggs (p. 90) may, however, probably be interpreted in this way, since THE SYNTHESIS OF NEW SUBSTANCES 385 the particles moved under these forces would seem to be comparable with those usually considered as microsomes. The role played by micro- somes in the synthetic activity of echinoderm embryos, and their relations with the mitochondria, have been discussed earher (p. 90). It will be seen that neither in Caspersson's nor in Brachet's theories is it supposed that the synthesis of proteins occurs actually at the gene inside the nucleus. If enzymes are sometimes produced in this way (p. 355), which is by no means certain, that can hardly be the general rule of pro- tein synthesis. One suggestion has been made (e.g. Wright 1945, Wad- dington 1939^), however, which minimises the difference between syn- thesis at the gene and synthesis in the cytoplasm. According to this, the genes directly produce substances which are replicas of themselves in most respects (or possibly in all except for the coimecting links which hold the genes together in the chromosome); and it is supposed that these repHcas pass into the cytoplasm and there control the synthetic processes. This hypothesis has the merit of simpHcity, but the evidence for it is slight; the fact that synthetic activity disappears or diminishes when the nucleus is removed suggests that the postulated gene-rephcas cannot at all fully take the place of true nuclear genes and it is therefore likely that they cannot actually be rephcas in the full sense of the word. It may very well be the case, however, that the immediate products of gene activity have a considerable chemical resemblance to the genes or to some part of them. Possibly the genes produce, as a first step, a product which resembles the protein which, in the chromosome, is combined with DNA, and this, passing into the cytoplasm, becomes associated with RNA, either near the nuclear membrane as Caspersson suggests or in the microsomes of Brachet. It will be seen from the discussion in this chapter that in spite of the very large effort which has been devoted recently to the attempt to dis- cover how genes operate, and the important body of interesting results which have been gathered, there has really been rather little progress towards the solution of the problem which has been put in the centre of the stage. This may be due to the inherent difficulties of the field; but the biologist who is not primarily a biochemist may be tempted to wonder whether the problem is not perhaps being envisaged in too simple terms. We have tended to think both of genes and of their products as definite and discrete particles, of, perhaps, the dimensions of a protein molecule or a little more. Possibly the active participants in gene-operations are actually of a higher order of complexity than this. As Goldschmidt has urged, and as we have seen above, there is considerable evidence which could be taken to support the idea that the active units in chromosomes 386 PRINCIPLES OF EMBRYOLOGY are relatively long structures, rather than 'point-genes'; and electron microscope studies on cytoplasmic structure (Fig. 17.2) suggest that we may have tended also to under-estimate the size and complexity of the active agents in protoplasm. We may, perhaps, find that it is necessary to think of gene action in terms not of enzymes or other protein molecules as we know them in solution, but of extended protein sheets and fibres, which may have properties which largely escape our present biochemical methods. Possibly it is in this way that we shall fmd an explanation both for the very large number of genes which affect the development of an organ and for the extreme precision with which they control it. SUGGESTED READING Bracket 1952^, b, Haldane 1954, Chapter 2, Goldschmidt 195 1, Muller 1947, Pontc- corvo I952fl, b, Spiegelman 1948, Schultz 1952, Wright 1945. CHAPTER XVm PLASMAGENES IN RECENT years a considerable body of evidence has accumulated for the existence in the cytoplasm of bodies of a more or less gene-hke nature. Geneticists have been particularly active in investigating them and have referred to them by a variety of names, such as plasmagenes, cytogenes, blastogenes, proviruses, etc. The first has been the most commonly accepted, and will be used here in a rather wide sense, to cover several rather different types of gene-hke entities. Broadly speaking, plasmagenes are revealed by two different kinds of evidence. On the one hand, breeding experiments may demonstrate that certain characters are inherited through the cytoplasm and not through the nucleus, and thus provide evidence of the existence of cytoplasmic hereditary determinants. Evidence of this kind can be of two grades. We may find that, throughout a number of generations, a certain character follows the transmission of cytoplasm, even when the whole set of chromosomal genes is removed by crossing. For instance, if a female of type A is crossed to a B male, and her female offspring again backcrossed to B males, and so on for several generations, the A chromosomes will gradually be replaced by B ones, and if any characteristics of A still remain in the offspring after several generations, one may conclude that they are dependent on hereditary factors transmitted through the egg cytoplasm and capable of continued multiphcation in the absence of their corre- sponding A genes. On the other hand, it may be found that although a character is transmitted only by a parent which contributes cytoplasm to the offspring, and is thus directly dependent on a cytoplasmic deter- minant, nevertheless this determinant can persist only in an organism pro- vided with the appropriate gene. In such cases (a good example is the 'killer' character in Paramecium) we have to do with a gene-dependent plasmagene. A different type of evidence for the existence of plasmagenes appears when it can be shown that a character can be transmitted from cell to cell by inoculation or other treatment with extracts which do not contain functional chromosomes; we may then conclude that we are confronted with a determinant, presumably derived from the cytoplasm, which can persist and impress some defuiite character onto the living cells into which it is introduced. The classical examples of such types of behaviour are the 387 388 PRINCIPLES OF EMBRYOLOGY transmissable viruses. When from such evidence we deduce the existence of a plasmagene, it is presumably implied that the cytoplasmic determin- ant is a fairly complex body, probably of the order of magnitude of a virus particle or a gene. Considerable caution should be exercised in making such deductions. Many years ago, in the early years of the investigation of cancer-producing viruses, it was pointed out that, given a tissue which has an appropriate competence, a particular type of cellular differentia- tion could be transmitted through an indefinite series of inoculations by means of cell-free extracts whose operative factors, however, were quite simple molecules which acted as evocators (cf Needham 1936/?). One knows now that the effective molecules might be even simpler than was reahsed at that time. It would be quite possible to carry on an indefinite series of transformations of gastrula ectoderm into neural tissue by means of inoculations of cell-free extracts, provided only that these extracts were sufficiently acid. Moreover, one might easily obtain phenomena which simulate a mutation of the virus. If the extracts came to contain free ammonia they would transform the gastrula ectoderm not into neural tissue but into derivatives of the axial mesoderm. More recently Lederberg (1952) has drawn attention to the same source of possible error. Again, Pollock (1953) has suggested a mechanism for the operation of self-perpetuating and even growing systems which might very easily be confused with plasmagcnes (Fig. 1 8.1). In the form he advances it, the idea depends on the phenomenon of enzymatic adaptation (p. 400) ; but somewhat similar systems might be produced in other ways. Pollock's suggestion is this; suppose that a substance A, supphed to a cell, causes it to synthesise an enzyme a which converts ^ to B; then suppose that B induces the formation of enzyme b, which converts B (perhaps in combination with other substances already in the cell) into C; then that similarly C is converted into something else, and that fmaUy a product is produced wliich is converted again into A. Such a system will be set going by the addition of an initial quantity of ^ and will then carry on indefinitely. In fact if the system absorbs energy, more A may be formed at the end of a cycle than entered it at the beginning, and the system will be able to grow. Such a system clearly has many of the properties attributed to plasmagenes; but it need not be incorporated in a particle. Thus to be justified in using grafting or inoculation experiments to postulate the existence of a plasmagene, one needs evidence not only that the character can be transmitted by cell-free extracts but that the effective factor in the extracts is a particle of the right order of complexity. Discussions which on the whole favour the importance of plasmagenes in development v^ be found in Darlington 1944, Darlington and Mather PLASMAGENES 389 1949, Spiegelman 1948, Medawar 1947, Ephrussi 1953 ; a somewhat more reserved attitude is taken by Brachet 19526, Beadle 1949, Waddington i94Sb, Sonneborn 1951a, &, and Haldane 1954. General reviews of the phenomena, less concerned with development, are Lederberg 1952, Caspari 1948, Cold Spring Harbor Symposia Nos. 11 and 16, 1948 and 1951, and the symposium pubhshed as Unites hiologiques donees de continuite genetique (Paris 1949). etc. Figure 18.1 A self-reproducing cyclic system. It is supposed that when A is' added to the cell, it induces the formation of enzyme a, which converts it, together with m from the rest of the cell, into B. After several such steps, during which raw materials (e.g. n) are used, and other substances (e.g. p, q) pro- duced, the system finally absorbs energy (Z) and results in the formation of more A than was originally supplied. This restarts the original cycle, and also brings into being a new cycle. (After Pollock 1953.) From the point of view of their possible importance in differentiation, plasmagenes may be considered under the following headings (c£ Fig. 18.2). I. Exogenons plasmagenes Many viruses, such as those producing disease, are clearly not essential constituents of the animal or plant and are introduced into the cell from outside. There is considerable variation in the ease with which this intro- duction can take place. Some of the bodies which were orginally thought of as true plasmagenes should be regarded as essentially exogenous factors for which infection is rather difficult. This probably appHes to the kappa particles in Paramecium (Sonneborn 1947, 195 1 which are also often considered to be plasmagenes, and which are char- acterised by the fact that they can arise anew within cells from which they were originally absent. Their initiation seems in all cases to depend on the functioning of corresponding genes in the nucleus and is impossible if the effective gene is absent. Other conditions of an environmental kind are usually necessary to bring the gene into play and cause it to produce the cytoplasmic factor. In Paramecium, besides the kappa particles which have already been described, there are cytoplasmic determinants of certain antigenic pro- perties (Sonneborn and Beale 1949, Beale 195 1, 1952, 1954). We have already (p. 3 59) referred to these in connection with the cytoplasmic control of gene activity and they also illustrate other points which may be relevant. Thus the cytoplasmic determinants (which are presumably particles, though they have not yet been seen) are under close control by genes. A given type of determinant cannot persist indefmitely in a cell from which the corresponding gene is absent; and in this their behaviour is perhaps similar to that of the Stentor kinetics. Further, the genes in the Paramecium nucleus appear to be able to bring into existence the cytoplasmic deter- minants which correspond to them, even if there were previously no representatives of this particular type in the cell. Thus if Paramecia are kept at 29-33° C. they will develop one of the D antigens (depending on PLASMAGENES 397 which of the D alleles is present in the nucleus). If the strain is then trans- ferred to 1 8°, the D antigen will eventually disappear, and an S antigen (corresponding to the S allele present) will take its place. It is, of course, difficult to be certain that there was absolutely no S antigen in the cells when they were originally kept at 29-33°, t)ut it certainly seems probable that this was the case, and that the cytoplasmic determinants producing the S antigens have been formed anew under the influence of the S gene when it became activated by the effect of the lower tempera- ture. These antigen-producing determinants therefore seem to provide examples of * gene-initiated plasmagenes'. Another important case is that described by Billingham and Medawar (1948, 1950, Medawar 1947, Fig. 18.6). If a patch of black skin from a spotted guinea-pig is transplanted into a white area, it is found that pig- mentation spreads out from the graft into the surrounding area, forming a sort of halo. In mammalian skin, pigment is formed as granules in the cytoplasm of a system of highly branched dendritic cells, the melanocytes, which are present even in white areas, although there the pigment- forming system is inoperative. Billingham and Medawar argue that the spreading of pigmentation which they have studied is due to the 'infection Figure 18.6 On the left, a shaved area of skin on a guinea-pig, into which a graft of black skin had been made 50 days previously: the black patch is due to induced pigmentation, most if not all of the grafted cells having died. On the right is shown the condition 20 days after an immunising graft from the same original donor was made ; the pigmentation is very largely bleached away, although some traces remain in the centre of the area. (After Billingham and Medawar 1950.) 398 PRINCIPLES OF EMBRYOLOGY of the melanocytes surrounding the graft by a virus- or plasmagene-like particle from the pigmented cells of the transplant. This particle must be capable both of self-duplication and of causing the host cell to produce pigment ; possibly it may be the pigment-forming particle itself, in which case these two actions would amount to the same thing. There are two alternative hypotheses to be eliminated before this theory can be accepted. On the one hand, the spreading of dark pigmentation around a graft might be due to the migration of actual pigmentary cells rather than of intra-cellular particles. Such migration certainly occurs in embryonic grafts of pigmentary cells in birds, but Billingham and Medawar argue that it does not account for all the phenomena observed in mammals, if indeed it occurs at all. In particular, it is difficult to recon- cile with the fact that if a graft of, say, black skin from the ear is grafted onto the sole of the foot, and pigmentation spreads around it, this black- ened area will have the characteristics usually found in sole-of-foot epi- dermis and not that of the ear-skin which initiated it. But although this makes it difficult to suppose that the spread of pigmentation is due to the migration of actual ear-skin cells, it suggests the other of the alternatives to the plasmagene hypothesis, namely that we are dealing merely with a serial evocation, in which some substance of low molecular weight diffuses from a dark cell to the neighbouring unpigmented one and sets going within it an already present pigment-producing system. There are, how- ever, phenomena which this possibility cannot easily explain. For instance, if a graft is made of black skin from guinea-pig A onto guinea-pig B, the halo of pigment formed round it retains the immunological specificity of individual A. If another graft of yl's skin is made onto B, it will set up an immunological reaction and as a consequence of this the pigment particles disappear from the zone of pigment spread around the original graft, although the dendritic cells themselves do not seem to be adversely affected. Billingham and Medawar, on these and other grounds, argue that we must be dealing with a particle large and complex enough to carry the immunological specificity of the individual from which it originated, and capable both of spreading from cell to cell of the dendritic system and of identical self-duplication. This case is of great interest and theoretical importance, since it is one of the very few examples of an alleged plasmagene in a higher animal which has been thoroughly studied in recent years, and in which there is no question of our being deceived by an extraneous virus. There are several further points to notice about it. First is the rather surprising fact that the 'plasmagene' carries the immunological specificity of the indi- vidual in which it arises, which makes it extremely difficult to transmit PLASMAGENES 399 from individual to individual (although this can be achieved if the graft is small enough not to provoke an intense anti-body formation by the host), hi this it is very different from most viruses. Secondly, the plasma- gene does not possess complete genetic continuity, but must arise anew during the development of the melanocytes from the neural crest; pre- sumably the initiation is gene-controlled, although the difficulty of inter- individual transplantation makes it difficult to prove this formally. Again, one must remember that the pigment-forming cells are peculiarly adapted to make contact w^ith their neighbours, and are known to pass pigment granules into cells, e.g. in the hair follicles, which are themselves incapable of manufacturing pigment. Thus the situation which enables us to recog- nise the existence of these plasmagenes by their transmission from cell to cell is a very unusual one. One might deduce from this that many such particles might exist in other cells, without our being able to detect them; equally one might argue that it is only because the melanocytes provide this peculiar mechanism of passing granules into contiguous cells that particles which can multiply and preserve their genetic character have been evolved to take advantage of it. Finally, the relations between the particles and the genetic factors in the nucleus remain obscure because we do not understand what genetic difference, if any, exists between a white and a dark area in a piebald guinea-pig ; both areas are, of course, derived from a single fertilised egg, and probably both contain exactly the same genes, although some authors have suggested that the piebald pattern is due to mutation of a colour-controlling gene in some of the somatic cells. It remains obscure, therefore, whether, when the pigment-forming plasma- gene is passed into a white cell and proceeds to multiply there, it is aided in doing so by the gene which initiated it, or whether that gene has mutated to an inactive form and the plasmagene is wholly responsible for the maintenance of its genetic character. Some suggestive but indirect evidence for the existence of cytoplasmic plasmagene-hke particles in normal cells can be found in some of the studies which have been made on tumour-inducing 'agents' of the kind which are often classed as viruses. These agents seem to be particulate in nature, and when suspensions of the particles are injected into appropriate healthy cells, they can multiply and cause the cells to develop into tumours. One important point in the present connection is that particles extremely similar to those of the tumour-virus can also be found in normal healthy cells; they are in fact the microsomes (Claude 1940). Further, when normal cells are acted on by certain carcinogenic chemical substances, the tumours which are induced are foimd to contain cytoplasmic particles capable of acting as tumour-viruses ; and it is certainly simplest to suppose that these 400 PRINCIPLES OF EMBRYOLOGY have originated by a comparatively slight alteration in the normal cell particles, to which one would therefore have to attribute a capacity for self-duplication. Finally, the tumour-viruses sometimes show extreme specificity in the type of tissue they can infect; indeed Rose [19 sib) has shown that a frog virus may be so specific that it travels through the body and settles in only one particular region of the skeleton (cf, the reference to regional specificity on p. 308). Now Rose has found that if the agent can be caused to grow in an unusual tissue, it may acquire a new specificity from it; its original specificity is sometimes retained, sometimes lost. He suggests that the new character may be picked up by an interchange process, akin to bacterial 'crossing-over' or 'transformation', with cyto- plasmic constituents of the normal cells which are essentially similar to the virus particles (but cf. Luria 1953). The suggestion is interesting, and considerable developments may be expected in this field; but at present the whole of this group of phenomena is so little understood that it seems dangerous to use it as a basis for a general theory of differentiation. For instance, according to Rose these tumour-inducing agents seem, after passage through a number of different hosts, to lose nearly all their tissue or even species specificity, which makes one wonder whether they may not be damaged forms of the normal cell microsomes, the damage being of such a kind as to render them insensitive to the normal control of the nucleus; if this were so, they could not be taken as evidence that the cyto- plasm of a healthy cell contains particles which are independent of the nucleus. Another type of phenomenon, which may involve the activity of plasmagenes, is that known as enzymatic adaptation (Reviews: Monod 1947, 1950, Monod and Cohn 1952, Spiegelman 1950, Gale and Davies 1953)- There is abundant evidence which strongly suggests (though perhaps it does not completely prove) that in bacteria, yeasts and other lowly organisms suitable for such studies, the formation of most enzymes attacking exogenous substrates is specifically increased by the presence of the substrate and in fact hardly occurs at all in its absence. The formation of the adaptative enzymes involves the synthesis of protein, and the physiology of the process has been studied by several authors (e.g. Spiegel- man and Sussman 1952). The formation of an adaptive enzyme in the presence of any particular substrate requires the activity of a corresponding nuclear gene, and we therefore have here a good example of the influence of substrates on the activities of genes. But it has been suggested that some- thing more is involved. Lindegren and Spiegelman [Cold Spring Harbor Syrrip. 1946) originally put forward the hypothesis that the production of the adaptive enzyme was a property of a cytoplasmic plasmagene, PLASMAGENES 40I which, in the presence of the substrate, could persist indefinitely even if the corresponding gene had been removed from the cell by crossing. Their original evidence, obtained in yeasts, w^as later shown to be inade- quate, but there still remains some evidence which may indicate that such plasmagenes exist (Spiegelman 195 1). Ephrussi (1953), (Ephrussi and Hottinguer 195 1) has also found evidence which suggests the existence of cytoplasmic self-duplicating particles concerned with enzymatic adaptation, hi his case, yeast cells cultivated in the presence of the nuclear poison acriflavine are shown often to give rise to colonies which grow abnormally slowly and in which the cells have lost certain respiratory enzymes and have an abnormal cytochrome system. The change is quite stable through many generations of vegetative division. Crossing experiments show that it is immediately dependent on a cytoplasmic, not on a nuclear, factor; but again further investigation has demonstrated that the plasmagene concerned is itself under the even- tual control of a gene. The enzyme changes induced by the acriflavine are not themselves adaptive, but they are closely similar to undoubtedly adaptive changes which yeast cells exhibit when cultivated in the absence of oxygen. It is questionable whether it is really appropriate to employ the word plasmagenes for any of the gene-initiated factors considered in this section. The character they share with the true plasmagenes is a certain ability to multiply in the cytoplasm. It is not clear, however, that any case is known in which a gene-initiated c}^toplasniic factor acquires complete autonomy in its powers of reproduction. Certainly the Paramecium antigen determin- ants can only persist for a very hmited period after the removal of the gene. The situation of the factors studied by Billingham and Medawar is obscure, since in the piebald guinea-pigs they studied, the originally colour- less cells into which the factor passes probably possess the same genotypic constitution as the coloured cells out of which it comes, the difference be- tween the cells being one which arises durmg differentiation rather than of a truly genetic nature. Beale (1954), who has studied these phenomena as closely as anyone, has recently expressed a lack of satisfaction with the term plasmagene for such factors. Haldane (1954) is apparently of a similar opinion and has suggested calling them mnemons. For con- venience in the present discussion, however, I shall continue to refer to them as gene-initiated plasmagenes. 5. The role of plasmagenes in differentiation It will be noticed that the overwhelming majority of the evidence for the existence of plasmagenes come from studies on micro-organisms. It 402 PRINCIPLES OF EMBRYOLOGY might be, however, that this is caused not by their rarity in other forms but by factors which make their detection particularly difficult. It is clear, for instance, that if plasmagenes were to play an important part in the differentiation of multi-cellular organisms, they could not in general be capable of easy infective transmission from one cell to another, since that would lead to an intermingling of different organs or types of tissue which should remain separate. Thus we camiot expect to fmd many cases similar to that of Billingham and Medawar, even if factors of an essentially similar nature are widespread. It is necessary, therefore, to approach the matter to some extent from an a priori point of view to try to determine how far plasmagene-like factors could fit in to the mechanisms of differentiation in so far as we understand them at present. It is clear that the exogenous factors mentioned under group (i) above do not come into the question. In the examples of the true plasmagenes mentioned in group (2), the cytoplasmic determinant is a part of the gener- al genetic constitution of the organism and no more directly related to the regionalisation of its various parts than are the nuclear genes. It is, however, possible to imagine that the cytoplasm of the egg of a given species might contain a number of different true plasmagenes localised in various regions. Each region of the egg would then contain characteristic cytoplasmic factors endowed with genetic continuity which might determine the nature of the organs which develop out of it. Such localised plasmagenes would, in fact, be the same thing as used to be referred to at the beginning of this century as organ-forming substances. Now there is no doubt that in many eggs different regions of the cytoplasm have different properties. The regions concerned are nowadays referred to as ooplasms, and opinion has rather moved against attributing their properties to the presence of substances which are autonomous over against the nucleus. The arguments which have swayed opinion against the old idea of organ-forming substances are numerous. One is that the evidence suggests that the ooplasms are only effective when they are able to interact with the nuclei. For instance, the cytoplasmic formation centre in the posterior of an insect egg only becomes active when nuclei reach it (p. 125) ; the same is true of the grey crescent ooplasm of the amphibian egg (p. 149). Again, differentiation from the egg to the final form takes place in a series of steps. It does not look as though we are dealing merely with the sorting out of a number of factors which from the beginning preserve their character un- changed, but rather as if development consists of a series of reactions dur- ing which the constituents of the system change continuously until the final condition is gradually built up. We are already faced with the difficulty of accounting for this progressive series of changes in a system PLASMAGENES 403 one of whose major components consists of genes which we beheve to retain their identity throughout. The difficulty is only made the greater if we have to suppose that the major factors in the cytoplasm also retain their identity. As a third argument, one may point to the fact that the localisation of different organs within the developing body may often be altered by factors which operate after the segregation of plasmagenes in the egg cyto- plasm must have been completed. For instance, one might be tempted to attribute the localisation of the organs in a developing Drosophila to the segregation of organ-forming substances or plasmagenes in the eggs, which are known to belong to the mosaic type; yet we have seen (p. 141) that environmental treatments apphed many hours after fertihsation can divert the differentiation of particular regions into abnormal paths. The mere fact that a gene, like aristopaedia, can cause a mass of tissue which should normally develop into an antenna to develop into a leo- instead, shows that even if we try to attribute the major process of differ- entiation to plasmagene-like bodies, these cannot be autonomous in their properties but must be highly susceptible to modifications caused by inter- action with genes. Finally, it would be still more difficult for a hypothesis which attributed differentiation to be activities of autonomous plasma- genes to account for metaplasia, which, though rare, does seem to occur (p. 308). It appears, therefore, that the postulation of true plasmagenes as organ- forming substances in the cytoplasm of the egg does not materially simpli- fy the theoretical task of accounting for the phenomena of differentiation. That does not necessarily mean, of course, that such bodies do not or can- not exist; we should have to take account of them if there was unequivocal evidence for the existence in the eggs of multi-cellular animals of cyto- plasmic factors which had genetic continuity independently of the nucleus. As yet there seems to be no compelling evidence to tliis effect, hideed, attempts to assess the autonomy of cytoplasmic factors in the egg over against the nucleus have been few and far between. The studies with hybrid merogons in Amphibia (p. 358) are perhaps the most promising. The facts there can probably all be accounted for in terms of a mere per- sistence of cytoplasmic character, without the need to postulate that the cytoplasmic factors can reproduce while retaining their specific nature. The case which argues most strongly in the opposite direction is perhaps that of Hadorn (1936), who found that epidermis derived from Tritums palmatHS cytoplasm fertilised by T. cristatus sperm developed the typical characteristics o£palmatus as late as after metamorphosis. This may indeed be evidence of the existence of a true plasmagene, but it might equally be 404 PRINCIPLES OF EMBRYOLOGY the result of a plasmagene initiated in the egg cytoplasm by the maternal genes during the maturation of the egg. One may conclude that there is hardly any evidence that plasmagenes with complete autonomous genetic continuity exist in metazoan eggs, and that it seems most improbable that the major phenomena of differentiation can be attributed to them. The same conclusion appHes even more forcibly to the plasmagenes of category (3), namely microscopically visible cytoplasmic particles with genetic continuity. These certainly occur in certain special cases, as for in- stance in ciliates, but in general the histological evidence makes it clear that differentiation does not consist to any large extent of the mere sorting out of the already existing visible particles in the egg cytoplasm. Such particles probably play an important part in development, but not by the mere retention of their original characteristics. The situation is rather different when we turn to the fourth category, that of gene-initiated plasmagenes. If these were to play an important part in development we should have to imagine that the various ooplasms of the egg differentially excite the nuclei which enter them; that the particu- lar genes which are activated in a given region then cause the appearance of cytoplasmic factors, and that these factors, when they have appeared, show a certain degree of autonomy, being able to reproduce for a short time with repetition of their character even if the nucleus is removed or changed. If one supposes that, once they have been formed, the autonomy of the plasmagenes is complete, this suggestion would come up against the same difficulties as confronted the hypothesis of organ-forming substances in accounting for the sequential character of differentiation and phenomena such as the metaplasia of retinal cells into lens in Wolffian regeneration. We have seen, however, that in the best-studied examples of gene-initiated plasmagenes the autonomy is by no means complete. If one waters it down sufficiently, the difficulties which have just been mentioned could be overcome. The hypothesis would then amount to the suggestion that during differ- entiation the genes cause the appearance in the cytoplasm of bodies with a certain limited amount of autonomy. There seems nothing impossible, or even very difficult, in such a suggestion. As was pointed out earlier (p. 212), Brachet (1944, 1952^) has argued with considerable persuasiveness for the importance of the ultra-centrifugable ribose-nucleic-acid-containing microsomes, and he does not hesitate to refer to these as plasmagene-like in character. The problem that still remains at issue is how far these parti- cles, once their character has been determined, become independent of the nucleus. Only the transplantation either of the nuclei or of the particles from one type of differentiating cell to another can settle the matter con- PLASMAGENES 405 clusively. There would be nothing surprising if experiment eventually showed that, in cells which are more or less completely determined and are in process of producing their final cytoplasmic constituents, the cytoplasm is able to carry on synthesising these nearly independently of the nucleus. However, such a fact does not yet seem to have been demon- strated. Whether, if it were, we should be justified in speaking of the effectiveness of plasmagenes in differentiation would be largely a matter of defmition; it would depend on whether we are satisfied that such gene- initiated cytoplasmic factors of limited autonomy are comparable to plasmagenes of the more completely autonomous kind. SUGGESTED READING Darlington 1944, Ephrussi 1953, Haldane 1954, Chapter 7, Medawar 1947, Monod 1947, Sonnebom 1951a or b, Weisz 195 1. CHAPTER XIX THE DIFFERENTIATING SYSTEM IT IS now time to try to formulate a general theory of differentiation based on the various factors which have been discussed above. Perhaps the first problem which should be considered is the develop- ment of differences between the various regions of the embryo. In the scheme of intra-cellular reactions which was suggested earlier (see Fig. 1 6. 1, p. 349), the nature of the cytoplasm affects the course of events at two different stages; on the one hand it provides the raw materials for gene activity and may thus differentially activate or inhibit different genes, and on the other it has the same relationship to the immediate gene products (and plasmagenes, if any). It is therefore easy to see how the constitution of the cytoplasm could set going a number of dissimilar processes of differentiation. In fact, it would be quite possible for this to occur through the interaction of the cytoplasm with the gene products, even if the activity of the genes themselves was exactly the same in all cells; but as we have seen there is actual evidence of nuclear differentiation in the various tissues, and there seems no reason to doubt that both the possible influences of the cytoplasm — on genes and on gene products — are effectively in operation. The next point is the existence o£ distinct alternative pathways of chemical change, leading to the production of a fmite number of defmite tissues ; and the pecuhar mixture of permanence and lability revealed in the phenomena of determination and modulation. The explanation for the distinctness of the developmental paths can probably be found in the nature of the cytoplasmic influences on the genes and gene products. All the different genes are made out of similar building blocks, i.e. the amino- acids and peptides which go to form the protein, and the nucleotides which form the nucleic acids. The same situation holds for the gene products. Thus we must suppose that the various genes (and gene products) will com- pete with one another for the available raw materials. There may also be other types of competition ; for instance a high concentration of one gene product A may inhibit the formation of another B, and so on. There will therefore be a situation of 'competitive interaction' in the formation of the gene products and another in their production of cytoplasmic sub- stances. 406 THE DIFFERENTIATING SYSTEM 4O7 Now in a system consisting of a mixture of raw materials for which several synthetic processes are competing, situations can easily arise in which a slight change in initial conditions will have a great effect on the fmal state; in fact, such systems will often be such that there are only a limited number of fmal states to which they can attain, the choice between one end-state or another depending on the concentrations of substances present at the beginning (Waddington I948l», 1954). To take a simple example of what may happen, consider two substances P and Q, which are being formed out of the raw materials A, B, and C, for the supplies for which they compete. Suppose competition occurs because P is formed from A and B, while Q is formed from B and C. Again for the sake of simphcity, let the reaction constants be the same for the two syntheses, as shown in Fig. 19. i; and let A, B and C diffuse into the system at rates proportional to the difference in their concentration inside (A, B, C) and outside {a, h, c), while P and Q are removed at rate ^3. Finally, let us suppose that the coupling of ^ and B to form P, and of B and C to form Q, are autocatalytic processes, i.e. are speeded up by the presence of already-formed P and Q. This is a simple form of a 'feed-back' mech- anism. The equations for the rates of change of the various components will be dA — - = k{a -A) - kPAB + J^2P2 at -^ = k(h-B)- kPAB + kP^ - hQBC + k^Q^ at Ac = k{c-C) - kQBC + k^Q" at dp -— = kPAB - hP' - kP dt ^ = kQBC - hQ' - kQ. At the steady state, we fmd a relation between P and Q of the form {khc + k\)P - {kha + k%)Q + kk{a -c) . . . (i) Now if ^3 is small compared with k (i.e. diffusion out of the system is slower than diffusion in), then we can neglect its higher powers, and we find P=^Q + f_^iZL_^ (,) c ko c 408 PRINCIPLES OF EMBRYOLOGY Thus if initially in a certain region the supply o£A is increased relative to that of C, we find that P will be increased relative to Q by something more than a proportionate amount, the excess being expressed by the last term on the right. And if the rate of removal of P, (that is k^), is greater than the rate at which P breaks down again into A and B, (that is kz), this excess can be considerable. We can also see from expression (2) that the exaggeration will be the more important the smaller the absolute values of P and Q; and these will also be reduced if ^3 is fairly large, so that P and Q are rapidly removed. ■*■ A ^ 0 — — *■ Figure 19.1 A system of competing chemical reactions, by which substances A, B and C become converted into P or Q. See text. (From Waddington 1954a.) Without going into further details, we can see that if two autocatalytic processes compete for raw materials, we may under some conditions find that an initial change in the supply of the materials produces an exagger- ated effect on the steady-state concentrations of the synthesised products, and thus on the rates at which these products can be made available out- side the system. If we suppose that .4, B, and C are the raw materials out of which two genes manufacture their immediate products P and Q, we have now deve- loped a picture by means of which we can see how a change in the con- centrations of these raw materials leads to exaggerated differences in the rate at which P and Q are passed out of the nucleus into the cytoplasm. It is, however, by no means the only model which might be appropri- ate. As Delbriick (1949) has suggested, there might be direct interactions between the two synthetic processes. These are perhaps most simply formulated by supposing that P is destroyed at some rate proportional to the concentration of Q (and vice versa). The equations for dPjdt and dQjdt will then contain terms in PQ. If we regard the system as closed. THE DIFFERENTIATING SYSTEM 4O9 rather than open as was the system discussed above, and if the suppHes of raw materials are taken as constant, the equations which result are of the same type as those which arise in the study of the growth of two popula- tions of animals which compete with one another for a limited food supply. Lotka (1934) had discussed the relatively simple situation of two populations (or substances) for which the equations take the form dp - = m^P- fe^P - k^,PQ 'p'^qp He shows that according as m^kq is greater or less than mjzpq, and nipkg, greater or less than mgkp, so the fmal state of the system is either wholly P or wholly Q, or a certain fixed ratio between them, or fmally the system is one which will finish up either entirely P or entirely Q according to the initial concentrations of these substances, Kostitzin (1937) has also discussed shortly the more general case in which there are many competing and interacting substances (or populations), so that we have a large series of simultaneous differential equations, each containing terms of the second order, such as P^ or PQ, etc. He shows that such a system may be expected to exhibit a number of alternative steady states, some at least of which are likely to be stable, and that the particular one which the system actually attains will in many cases depend on the initial conditions. Competitive interactions are not only almost necessary consequences of the nature of the situation, but there is definite and direct evidence for their existence. Perhaps the most striking is that produced by Spiegelman (1948, 1950) from the study of adaptive enzyme formation in yeasts. If a yeast is grown in the presence of two substrates, for neither of which it originally possesses the appropriate enzyme, it will gradually manufacture the suitable adaptive enzymes, which are of course protein in nature. If the supphes of nitrogen are restricted, these two proteins enter into obvious competition with one another for it, one or both of the enzymes being formed more slowly when the yeast is adapting to two substrates simultaneously than when there is only one new substrate (Fig. 19.2). Spiegelman also shows that the systems producing the adaptive enzymes are self-reinforcing or 'autocatalytic' ; and the way in which a muscle cell, for instance, fills with myosin, suggests that the same is true of the formation of many cytoplasmic proteins. We have thus all the components 410 PRINCIPLES OF EMBRYOLOGY required for a system which will tend towards a limited number of alternative end states. It is not so easy to give an actual example of a system in which the alternative end states depend on specific inhibitions, of the kind postulated by Delbriick. However, Horowitz (195 1) has mentioned a case, which, although it is still somewhat hypothetical, may serve as an example of the kind of situation which it would be most desirable to analyse. When the mould Neurospora is grown in sulphur-deficient media it develops Simultaneous adaptation maltose and galaaoK D 240 360 Minutes Figure 19.2 Simultaneous adaptation of yeast to maltose and galactose. The curve B shows the activity in fermenting galactose, when that is the only sugar present; D is the corresponding curve for maltose. When both sugars are present, the splitting of galactose is not much affected {A) but the growth in maltose-splitting activity is competitively inhibited (C). (After Speigelman 1948.) the enzyme tyrosinase, and tends to become blackened with melanin pigment if any suitable substrate is available. It seems probable that in fact this enzyme is also produced in normal media containing sulphur, but that in them some sulphur-containing compound which inliibits tyro- sinase activity is also formed. Horowitz asks what would happen if one of the products of tyrosinase activity could combine with the inhibitor and destroy it, or in some other way prevent its appearance. Then, he suggests, if a mould were grown for some time in a sulphur-deficient medium, it would acquire a high content of tyrosinase, and on transference to a normal medium this enzyme would continue to be active unless or until the production of inhibitor could overtake its inactivation by the tyro- sinase-system. We might then have a very simple example of a system THE DIFFERENTIATING SYSTEM 4II with two alternative types of differentiation (with and without active tyrosinase and formation of melanin pigment), the choice between which would be dependent on the previous history of the strain (whether it had spent a period in a sulphur-deficient medium). There appears therefore to be no difficulty in accounting in one or other of these ways for the existence of distinct alternative paths of differentiation. Within a given path, there are two types of phenomenon for which we have to provide an explanation. The first is the existence of a range of variation of the kind which is exhibited in 'field' processes. For instance, if some cells in the limb region enter on the path of develop- ment leading to cartilage and bone formation, they may have the character of the femur or, on the other hand, of some other part of the limb skeleton. A somewhat similar phenomenon is that of 'modulation'. In this case a given tissue assumes a range of histological forms in dependence on the nature of its environment, but throughout all such changes retains its essential character unimpaired. Presumably in both cases the variations are basically quantitative in nature, and indicate that the concentrations of the reacting gene-products and cytoplasmic substances can take a certain range of values while still remaining within one and the same alternative path of development. In contrast to such flexibility of behaviour is the essential permanence which is alluded to by speaking of tissues as 'determined'. When tissues are modulated by some external influence, they nevertheless retain their original character and can re-express it when suitable conditions arise. Can the hypothesis which attributes determination to competitive inter- action provide a satisfactory explanation of this ? The question has been little studied, either theoretically or practically. There is no doubt that one can invent systems of competitive interaction which would, if they actually existed, make differentiation very difficult to reverse. The mere co-existence of numerous interacting substances would almost suffice. Evolutionary changes hardly ever in practice get reversed, just because they depend on such numerous gene changes that it is extremely improb- able that all the reversals will happen simultaneously. In the same way, a highly complex system of competitive interactions would scarcely ever be brought to retrace its steps. But that is a very generalised argument; and the occurrence of modulation, and of pecuHar combinations of lability and fixity of character (such as the labihty of avian epidermis which allows it to be induced to form a feather, combined with its fixity of tract-specificity, p. 259), make one wish for some rather more detailed understanding. This could probably come partly from a mathematical investigation of the theory of stabihty of competitively interacting systems. 412 PRINCIPLES OF EMBRYOLOGY Much could probably be learnt, also, from specially designed experiments on simple examples of them. Until such time as further theoretical or experimental studies have been made of the properties of such systems, one is left with a certain freedom of choice as to what hypothesis seems adequate to explain the facts of determination and modulation. It may well be that there is no need to assume anything more than competitive interaction between autocata- lytic processes leading from genes to gene products, and from the latter to the fmal cytoplasmic constituents. Some authors, however, feel that a further factor tending towards persistence of character is required, and suggest that this can be found by invoking the presence of plasmagenes. This possibility was discussed in the last chapter, where the conclusion was reached that gene-initiated plasmagenes would seem the most likely kind to play a large role in development, but that although there is no doubt that the cytoplasm contains complex bodies of a roughly gene-like order, one requires more evidence of their independence of nuclear control before accepting them as plasmagenes. This additional evidence can, as far as one can see at present, be sought only in a further study of the properties of the micro- somes to which Brachet has attached so much importance in the synthesis of proteins. Are they indeed the main site of protein production, as he suggests? And if so, have they in addition the property of multiplying and retaining their own specific character in some degree of independence of the genes? If so, one might allow that they were plasmagenes of one or other of the types indicated in Fig. 18.2, Really conclusive evidence on these points will, probably, only become available when the technical difficulties of transplanting microsomes from one cell to the other are overcome, and their degree of autonomy over against the nucleus can thus be investigated. In the meantime, the best evidence we have as to the developmental functions of microsomes comes from the phenomena of evocation. In evocation we are undoubtedly confronted with a situation in which an environmental influence, impinging in the first place on the cytoplasm of the competent cell, causes it to adopt one or other of the alternative paths of development open to it. The fact that the reacting tissue retains its own specific characteristics — T. alpestris ectoderm forming typical alpestris neural tissue even if evocated by T. cristatus mesoderm — shows that the developmental paths are under genetic control and that the evocation involves the differential activation of a particular set of genes. The problem of the mechanism of evocation therefore becomes that of the nature of the influences which can activate or inhibit genes; that is to THE DIFFERENTIATING SYSTEM 413 say, which can produce crucial changes in the systems of competitive interactions. We know that the initial evocating stimulus may be com- paratively sHght, even a mere change in pH. We do not yet know on exactly what part of the cell system this obtains its effect. Weiss (1947, 1949^) has pointed out that an evocating stimulus might act at the cell surface, by causing the accumulation there of a particular molecular species, with a consequent depletion of the deeper parts of the cell and an alteration in the 'molecular ecology', or systems of competitive inter- action (Fig. 19.3). There is, however, little convincing evidence that evocating actions take place primarily at the cell surface (cf p. 213). \ T> J Figure 19.3 Diagram showing how two different substances (upper and lower rows) acting on the surface of a cell might attract to the surface different specific internal constituents, and thus cause progressive cellular differentiation. A conceivable mechanism for evocation. (From Weiss 19506.) Indeed the most critical evidence as to the location of evocator action speaks in the opposite sense. Waddington and Goodhart (1949) in- vestigated the position taken up within the cell by the sterol-hke hydro- carbons which are extremely active evocators. It was found that they are not absorbed on the surface or on the nucleus, but on lipo-protein granules (lipochondria) in the cytoplasm, which then break down to give microsomes. 414 PRINCIPLES OF EMBRYOLOGY This is the only case in which the location of an evocating substance within the cell has been verified; and the trail appears to lead to cyto- plasmic particles and eventually to the microsomes. Brachet (1944, 1947, 1952) has advanced a number of lines of argument suggesting that these particles are intimately involved in evocation (p. 212). It may be that all or most of the many substances capable of evocating neural tissue act in the first place on these granules. We saw, however (p. 222), that the microsomes are only one element, though an important one, in the com- plex flux of cellular metabolism, and that one cannot consider the mechanism of evocation without taking account of gene action, protein synthesis, respiration, etc. The biochemical processes symbolised by the two cycles of reactions in Fig. 16.1, from the cytoplasm to the genes, and from the cytoplasm to the gene products, must actually involve all the basic metabolism of the cell. It is fruitless to envisage evocation as a special reaction, carried out by some particular part of the cell which can be, in theory, isolated from the general body of the living system. We must be prepared, therefore, to^md that several different types of influence, im- pinging on the double cycle of cellular metabolism, may result in the same effect of swinging these cycles into one or other of the alternative modes open to them. Similarly, when we think of particular groups of genes being activated in different tissues, the cytoplasmic conditions responsible for this probably cannot be reduced to the mere concentration of certain inactive raw materials which lie quietly there for the genes to utilise. The 'substrates', for which we have envisaged the genes competing, will themselves in many cases be involved in active chemical processes of respiration, energy transfer, etc. The genes and gene products must be thought of as focal points in a continuously active and dynamic system. SUGGESTED READING Horowitz 195 1, Lehmann 1950, Schultz 1952. CHAPTER XX INDIVIDUATION— THE FORMATION OF PATTERN AND SHAPE DIFFERENTIATION does not consist merely in the production of new 'substances' — be they simple ones, such as pigments, or complex, such as the various types of tissue. We also have to consider the arrangement of these substances into definite relative positions, and, usually, the moulding of them into characteristic shapes. Development, in fact, produces not merely tissues but organs. It is to this type of phenomenon that the name 'individuation' has been given. It has, as has been impHed above, two rather different aspects. On the one hand there is the question of the spatial distribution of the different substances. For instance, within the sheet of developing mesoderm a notochord develops in the dorsal mid- line, flanked on either side by somites, with the nephric mesoderm more laterally again and side-plate mesoderm outside that. We have earlier (p. 12) used the term 'regionalisation' to refer to the appearance of different parts within an originally uniform expanse of tissue. This is one of the aspects of individuation and before considering it further we should remind ourselves that regionalisation normally takes place so as to produce definite patterns of arrangement of the different parts. It is not adequate to picture it merely as a process by which a number of intermingled entities become sorted out into heaps of like components ; we must add the fact that the heaps are mutually arranged in orderly patterns. The other aspect of individuation is the formation of three-dimensional structures. For instance, the hollow sphere of the blastula undergoes the process of gastrulation and thus acquires a new and definite con- figuration; or again, a neural plate rolls up into a neural tube, which is characterised by the well-defined swellings of the brain vesicles, etc. AH such processes are 'morphogenesis' in the strict sense, since that word really means the development of shape. The shapes of organs and of the body as a whole continue to change throughout most of life owing to the unequal growth of different parts. Such processes of relative growth have been considered in Chapter XIII; they may be considered as secondary morphogenesis. What we shall be concerned with in this chapter are the processes of primary morphogenesis, by which the original shape of the organ rudiments is first brought into being. (The distinction between these two categories of primary and secondary morphogenesis is not 415 g PRINCIPLES OF EMBRYOLOGY sharp but k B a useful rough classification and we shall see that in primary morphogenesis there are many factors wUch play a more important part than di6ferences in growth rate.) The two aspects of individuation-morphogenesis and pattern torma- tion-are obviously closely coimected with one another. It is hardly to be supposed that any comphcated three-dimensional structure will devdop uiJess the material out of which it is made has a ready developed a pattern of different properties m its various parts. Thus some degree o pattern formation probably always precedes any but the very sm.plest Lorphogenetic processes. Contrariwise it is to be expected that a develop- ing pattern will be influenced by the shape of the area or mass m which it is forming and we shall find examples which demonstrate that ths is the case. It is, however, helpful to use the distmction between pattern formation and morphogenesis as a means of arranging the subjects which require discussion into some sort of order. Moreover there is a certam difference in the kind of processes which must be involved m the two classes of phenomena. Pattern formation can, and frequently does, go on within a mass whose overall shape does not change. It requires the postulation of forces of an essentially chemical or physico-chemical order -diffusion, facilitated synthesis and the like. Morphogenesis, on the other hand involves the actual movement of masses from one spatial position to another, and requires the intervention of physical forces such as those of surface tension, attraction, contraction, expansion etc. Pattern formation and morphogenesis are typical examples of field phenomena, since they mvolve processes which are both extended throughout a region of space and which also have a certam unity. As was suggested earher (p. 23) such fields arise from the interaction or a number of different factors each of which is extended throughout the region involved. We cannot expect, therefore, to be able to attribute the forma- tion of a pattern to the action of any one single factor, but must expect always to fmd that several different things are involved m it; and tlie same expectation of a multiplicity of causes rather than a single cause holds goods for morphogenesis. ■ . j-ff ^ Although pattern formation and morphogenesis occur m the differen- tiation of all organs and embryos yet there are not very many instances in which they have been closely studied as the main subjects of investigation, and we still know disappointingly little about the nature of the factors involved in them. What we do know suggests that the operative factors are very different in different cases. The shape of a mass of tissue, for m- stance, may in one case be altered by changes m the tension m the surface of the mass, or again by changes m the adhesiveness of the cell membranes, INDIVIDUATION — FORMATION OF PATTERN AND SHAPE 417 by differential imbibition of water, by reaction to pH gradients, by differential adhesion to other neighbouring cell masses, by the move- ments of its individual constituent cells, and probably in many other ways. It seems impossible to hope that we shall ever discover any single basic mechanism of pattern formation or morphogenesis, as we may still hope to fmd, for instance in the mechanism of protein synthesis and its control by genes, the fundamental mechanism for substantive differentiation. In discussing pattern formation and morphogenesis, therefore, one can hardly hope to do more than provide a number of illustrations of the general nature of the processes which are at work. I. Primary and secondary expressions of pattern Many of the most striking animal patterns which we can observe are probably secondary or derived expressions of the underlying primary pattern, and it is the formation of the latter rather than that of the visible configuration derived from it which presents the really interesting prob- lem. An example will make the distinction clear. If certain hormones (thyroxin or oestrone) are injected into fowls of certain breeds a change occurs in the colour of those parts of the feathers which are being formed while the hormone content of the blood is at a high level. Lillie and Juhn (1932) studied the shape of the coloured region which is produced in response to single doses of various sizes. They showed that the tlireshold of hormone concentration, which has to be surpassed before a colour alteration is produced, is lowest near the rachis of the feather, and rises towards the sides. They also came to the conclusion that the various parts of the feather differ in the time-lag which has to elapse between the attaiimient of the hormone threshold and the actual deposition of altered pigment. On the basis of these two variables it is easy to see that one might obtain a pattern consisting of a single spot near the rachis, by making an injection which did not raise the concentration as high as the threshold of the lateral parts of the feather. If, however, the injected dose were larger, so that it surpassed the threshold even of the lateral parts, some form of transverse bar would be obtained. The shape of this bar would depend on the rela- tion between the time taken for the hormone level to fall again by excre- tion and the time-lags of the various parts of the feather. Indeed, if the hormone were excreted very rapidly the central parts of the feather, with their long time-lag, might fail to respond at all, although the concentra- tion had for a short time reached the necessary threshold and had produced an effect on the more quickly-reacting lateral parts of the feather. This would give rise to a pattern consisting of two spots near the edges. Thus 4l8 PRINCIPLES OF EMBRYOLOGY according to the quantity of hormone injected and the rate of its ex- cretion, quite different visible patterns might result; but they are all secondary consequences of the thresholds and time-lags which vary in a defmite manner within the feather. It is the arrangement of these varia- tions which must be regarded as the primary pattern. It is comparatively easy to understand how, once the primary pattern is given, various mani- festations of it can be made visible by treatments such as hormone injections. The fundamental problem is to understand how the time-lags, thresholds, etc. come to be arranged in a pattern in the first place. (It should be mentioned that the 'classical' story of hormone-induced feather patterns, given above, has been severely criticised by 'Espinasse [1939]. It has been quoted here because it brings out very clearly the distinction between a primary pattern and the derived expressions of it.) 2. The origination of pattern One of the most thorough investigations of formation of primary patterns has been concerned with the coloration of the wings of Lepidop- tera (Reviews: Henke, 1935, 1948). The comparative study of nearly related species has made it possible to distinguish a number of different elements, or systems of elements, which are combined together to form the pattern of any particular wing. In the most general form there are three such systems, which in Henke's terminology are referred to as 'fields' ; first, a general field, which can be thought of as covering the whole wing; second, enclosed within this is a peripheral field; and third, enclosed within that again, a central field. Both the central field and the peripheral field usually stretch right across the wing from its anterior to its posterior margin so that the general field occurs only at the base and at the lateral edge. There are usually strongly marked features at the boundaries between the fields forming a series of lines (the 'Querbinden' or transverse bands) (Fig. 20.1). These three main fields seem to be epigenetically more or less indepen- dent. They can be very differently developed in different species. Quite often the peripheral field extends right down to the base of the wing, so that the general field disappears in this region and occurs only at the distal edge of the wing. Moreover, experiment shows that different fields are determined at different times during the development of the wing-bud. If small wounds are made by cauterisation in the developing bud after a given element in the pattern is determined, the resulting wing will show the normal pattern disturbed only by the presence of the dead area (Fig. 20.1). If, on the other hand, at the time of operation the pattern was not fully determined, its development will be modified in some way INDIVIDUATION— FORMATION OF PATTERN AND SHAPE 419 (as we shall see later, this modification may take the form of an arrest of the pattern at an early stage in its development). If cauterisations are made at different stages it is found that some elements of the pattern may act as though they were fuUy determined at a time at which others are stiU capable of bemg modified. We have therefore to consider each of the fields as representing an independent unit within which pattern formation is proceeding. This means that the lepidopteran wings are rather compli- Ptriphtral ,.' ,^"^ field y' I ^^nlraX y I held Figure 20.1 On left: generalised scheme for the three main elements in the wing pattern in butterflies G/indicates the maximum extent of the General Field P/" of the Peripheral Field (= 'Umfeld'), and C/of the Central Field ('Zentralfeld') On right: the results of cauterising the pupal wing at various stages. In the graph, age at operation is given (in days after pupation) as abscissa ; as ordin- ate IS shown the percentage of cases in which the Peripheral Field or the Central Field behave in a mosaic manner which shows that they have already been determmed. The three wings drawn on the graph illustrate the results ot an early operation, at a time when only the spots of the edge (General Field) are determmed then a case in which the Peripheral Field is determin- ed but the Central Field not, and finally a case in which the whole pattern is determined. (After Henke 1948.) cated examples of pattern, since they consist of a number of different mdependent areas rather than a single one; but this complexity is com- pensated for by the great variety of patterns which are available in differ- ent species and the ease with which they can be studied. Henke has discussed the different types of pattern which might theore- tically be expected to form within any one area (Fig. 20.2). Before any pattern appears one must imagine that the area is more or less homogen- eous. The simplest pattern would be a random distribution of spots of various sizes, the frequency of the different sizes falling into a normal distribution. This he caUs a 'spatter' pattern. The next class he distinguishes IS that m which some sign of periodicity or rhythm can be seen- for 420 PRINCIPLES OF EMBRYOLOGY instance, by the formation of spots of more or less similar size lying at roughly equal distances from one another, or a series of Hnes at approxi- mately equal distances apart. These he speaks of as 'simultaneous rhythms', distinguishing them in this way from other rhythmic patterns which involve time, which v^dll be mentioned later. The simultaneous rhythm may be on a small scale in relation to the whole area covered, in which m;£ Figure 20.2 Types of pattern, according to Henke, illustrated in lepidopteran wings: a, spatter pattern; h, c, d, e, 'simultaneous rhythms' of increasing wavelength, leading to the case/ when there is only a single element present; ^, a single spot which has a transitional zone around it; ^, a diffusion field; i, accumula- tion of diffusing material at the boundar)'; k, I, m, are other examples of boundary accumulations in more complex patterns; n, a 'centric rhythm' resulting from a Liesegang-like phenomenon in a diffusion field; 0, p, q, successive diffusion fields ; r, s, interactions of the diffusing substance with the surroundings. (After Henke 1948). case the area will include a number of elements of the pattern. Alterna- tively, if the periodicity is on a larger scale, there may be few, or in an extreme case only one, repetition of the basic element. Henke seems to irrtply that such rhythmic or periodic patterns can arise spontaneously within an originally uniform region. He suggests (1948) that the periodicity arises through some form of competition, pre- sumably for diffusable substrate materials, between spots which were originally irregular in size and in disposition. A more precise account of how such regular patterns can arise has been provided by Turing (1952). We shall return to this later. Henke then considers a number of other types of pattern which arise in INDIVIDUATION— FORMATION OF PATTERN AND SHAPE 421 a more complicated way. For instance, each spot in a simultaneous rhyth- mic pattern may have not a sharp boundary but one w^hich grades gradually into the background; or there may be a reaction between the spot and the background in such a way as to outhne its periphery. Again, from a single spot or elongated area some substance may diffuse outwards and give rise to a periodic pattern as a consequence of the well-known (though little understood) Liesegang phenomenon. This form of rhythmic pattern Henke refers to as a diffusion rhythm. Mere inspection is some- times sufficient to suggest that a given rhythmic pattern belongs to the simultaneous or the diffusion type. In the former one would expect the pattern to be rather irregular but to show no indication of any change in the wave-length, whereas in the latter one expects a more precise forma- tion of the pattern and a gradual increase or decrease in wave-length. A final distinction of the two types can, however, only be reached experi- mentally. In a number of cases, indeed, experiment has demonstrated that diffu- sion plays an important part in the production of a certain pattern. For instance, in the wings shown in Fig. 20.3 the central field originates from two pomts, one on the anterior margin of the wing and the other on the posterior. From these, two streams of some substance spread across the surface of the wing until they meet in the middle, as is shown by the fact that if the wing is wounded by cauterisation during the period when the C37. ^:d. ck c» ^cm Figure 20.3 Diffusion as the process producing the Central Field in the wing of the meal- worm Ephestia. The upper row show examples of inhibition of the diffusion by cauterisations performed at the time when it would normally be pro- ceeding (2- to 3-day pupa). In the middle row, the cauterisations were made earlier; the diffusing material has merely by-passed the necrotic areas. In the lowest row, a shows the normal extent of the central field; b a case when It IS narrowed by the action of a gene 5y; c, a similar narrowing caused by high-temperature treatment during the diffusion; d, a widened tield produced by the gene Syb; e, a similar effect caused by early hich- temperature treatment. (From Henke 1935, after Kiihn and v. Engelhardt ) 422 PRINCIPLES OF EMBRYOLOGY spread should be taking place the process is brought to a standstill, and the intermediate stages of the diffusion are revealed. Henke calls patterns formed in this way 'spreading fields'. The edges of such fields may often be outlined by some product of a reaction between the substances charac- teristic of the two areas. How far does this account of some fundamental types of pattern forma- tion, which was primarily based on the analysis of lepidopteran wings, provide us with an explanation of the kind of phenomena which we meet in the development of embryonic organs in general? It is obvious that a step of the greatest importance would have been taken if we had reached an adequate understanding of the spontaneous appearance of rhythmic or periodic patterns within an originally uniform area. Henke rather vaguely suggests that this may be due to competition between originally irregular spots. Turing (1952) in a paper with the challenging title of 'The Chemical Basis of Morphogenesis', has elaborated a mechanism by which a regular pattern might arise witliin a completely homogeneous system. He considers a region (imagine a plane two-dimensional area to make it simpler) in which a number of chemical reactions are proceeding. If these interact with one another by involving the same substances, or by producing products which act as catalysts or affect the rates of other re- actions in any way, then the straightforward situation would be the attainment of some sort of balanced equihbrium condition throughout the whole area. But such an equihbrium is only a statistical phenomenon; actually the system will be disturbed by shght chance variations from place to place. Now it is easy to imagine special systems of reactions such that the equilibrium is unstable; if by chance one substance appears at a certain place in slightly too high a concentration, it will go on increasing. From each such 'high' spot, the substance will diffuse outwards, so that the spots will gradually enlarge. Turing has set up mathematical equations for such systems, and, choosing some arbitrary figures to express the rates of the reactions and of the diffusions, has solved them by means of a modern computing machine. He found that under certain conditions one might expect to get a pattern of a few fairly large areas or irregular blotches of high values of some particular substance. Moreover in some circumstances, the pattern might be more regular, showing a rhythm Mdth a definite wave-length dependent on the physical and chemical magnitudes controlling the reactions. Turing compares his 'chemical wave-length' with the interval between regularly appearing structures in an animal or plant. For instance, if the circumference of the cyhndrical body of a Hydra were just about six times the wave-length, one might attribute the animal's hexagonal symmetry, INDIVIDUATION— FORMATION OF PATTERN AND SHAPE 423 and the appearance of six tentacles, to such a mechanism. But tliis does not seem very convincing. One of the most important characteristics of embryonic development is that the patterns which arise tend to be accom- modated to the total amount of material available. For instance, a normal embryo can be formed from half an egg, or from two eggs fused together; or if a flatworm is cut longitudinally into strips each much narrower than the normal breadth of the worm, nevertheless each of these will regenerate a complete head. In patterns which behave in this way, the distance apart of the various elements cannot be fixed by any defmite chemical wave-length dependent on the unchanging values of rate con- stants, diffusion constants, etc. Rather the pattern must arise as a whole within the boundaries of the material available. The simultaneous rhythms of Henke or the chemical rhythms of Turing cannot, then, provide a general explanation of the periodic patterns which are important to animal morphology. It seems in any case improbable that fundamental rhythmic patterns, such as those of the somites of the vertebrate body, would be dependent on such an inherently chancy mechanism as that investigated by Turing. Probably the processes which he and Henke have discussed play a part only in the quasi-periodic dapphngs and mottlings which often fill up relatively unimportant spaces. The fact that a developmental pattern is usually found to become either enlarged or diminished in scale so as to fit into the available material sug- gests that the boundaries of the mass of substance play a major part in the processes by which the pattern is formed. For instance, in a regeneration blastema of a flatworm, it may be that some process always attains a critical value in the midline and falls off to zero at the two lateral edges. If this, or something like it,, were the case, one could understand how a complete head appears even on a very narrow strip. Again, in Henke's wings which are characterised by a central field, we may suppose that the position on the anterior margin from which the diffusion of the field starts is always midway between the base and the tip, however large or small the wing may be. It is only by some such relations as this, in which the pattern is produced in dependence on the boundaries of the material available, that the facts as they are observed can be adequately under- stood. Although, as has been stated above, developmental patterns often retain their completeness even when the material available is considerably greater or less than normal, this is not always the case. The relation between pattern and mass is certainly not simple and probably differs in different cases. A number of other instances will be mentioned below. 424 PRINCIPLES OF EMBRYOLOGY 3. Some actual patterns It is now time to turn from this somewhat abstract discussion of the fundamental principles of pattern formation to the consideration of one or two actual examples of patterns which have been relatively fuUy analysed. Studies employing essentially biological methods (e.g. investi- gation of the development of mutant types, or the performance of surgical operations) have shown that many apparently simple patterns result from the interplay of numerous factors. Investigations by chemical or physical methods have not as yet progressed nearly so far. Indeed in most cases we have no actual evidence at all as to the physico-chemical nature of the processes involved and can hardly proceed beyond such a priori arguments as those discussed above. (a) Drosophila wing venation It is, as might be expected, in Drosophila that genetic methods have provided us with the greatest mass of information concerning patterns. Most of the important principles which emerge from such studies can be illustrated by a consideration of the venation of the wings (Waddington 1940b). The veins of the adult wing present a fairly simple pattern, con- sisting of five.longitudinal veins running from base to tip, with two cross- veins, an anterior one between L3 and L4 and a posterior between L4 and L5. This pattern arises in a series of stages, of which we may distingmsh three: (i) The prepupal stage, in which L2 is absent, L3 and L4 are umted from the base to near the middle of the wing blade, there is a marginal sinus right round the edge (part of which corresponds to the later Li) and there are no cross-veins. At the end of this stage the wing is inflated mto a balloon-like shape by the pressure of the internal fluid and all visible traces of the prepupal venations disappear. (2) When the wing contracts again, in the pupal period, the five longitudinal veins make their appear- ance. (3) In a sHghtly later phase, at the very end of the contraction, the two cross-veins appear (Fig. 15. i, p. 33 1)- Some genes have effects which affect the five-rayed pattern of the longitudinal veins as a single organised unit (Fig. 20.4). The effects of these generaUy acting genes are, however, of several different kinds. Perhaps the most striking, but the least illuminating with regard to pattern forma- tion, are genes such as dumpy which, by affecting the pupal contraction, distort the pattern of vehis after it has been laid down. This is not really an effect on pattern formation, but only on the expression of the Pattern. Some other effects, however, are more radical. For instance, in shifted ^W the veins from L2 to L5 appear as though squeezed together, divergmg at a lesser angle. This occurs without any noticeable change m the outUne INDIVIDUATION— FORMATION OF PATTERN AND SHAPE 425 of the wing; the effect on the pattern formation must be brought about by altering the reaction of the material to effects of the wing boundary, rather than by altering the boundary itself. Other cases, however, suggest that the amount and shape of the material available to form the wing may have an effect on the pattern of venation. For instance, in dachs the longitudinal veins are splayed apart and at the same time the wing is Figure 204 ^^.^^-'^ontrolled modifications of the pattern of wing venation in Droso- phila.Ina, h, c, d, e the mutant wing is drawn in a fiillUne, superposed on a dotted drawing of a normal wing; a, in shifted-2 the longitudinal veins are pinched together; h, in broad, the wing blade is relatively broader than nor- mal and the veins diverge at a greater angle; c, veinlet may remove a con- siderable part of the distal region of the veins, and the posterior cross-vein IS moved to fit; d, cubitus ititerruptus removes the distal part of the fourth vem, and the fifth then shifts upwards ; e, dachs produces a short square wing, m which the veins diverge at a greater angle than normal;/, the dachsous wmg IS large and the veins diverge at a large angle, notice also the cross- veins; g, much-altered venation when the wing shape is highly abnormal {dachsous-fourjointed-plexus); h, small mirror-image twin wing produced by blot. (After Waddington 1940, etc.) 426 PRINCIPLES OF EMBRYOLOGY square in shape, and it seems probable that it is the change in wing shape which has brought about the akeration in the laying down of the pattern. It is not quite clear what happens to the pattern if the size of the wing is reduced, without alteration of its shape, before the venation is deter- mined; genes which produce small wings (such as miniature) act largely, if not wholly, after the pattern has been laid down and thus, like dumpy, distort something which is already in existence rather than alter the con- ditions under which it comes into being. Genes are known, however, which cause increases in the wing mass earher than the period of pattern formation. One is dachsous; this usually produces a fairly slight increase in size and a five-rayed pattern of longitudinal veins appears, the angle of divergence being, of course, larger than usual. In hlot, on the other hand, the exaggeration in size is greater, and extra longitudinal veins make their appearance. In extreme cases these extra veins can be seen to form a mirror-image of the normal venation, the mirror plane being along the position of L5. Such duplication and mirror-imaging is rather common when a pattern is developing in a mass of tissue which is, as it were, too large for it. One feels that it should offer an important clue as to the nature of the essential processes concerned in pattern formation, but so far no one has suggested just how it should be interpreted: stimulating discus- sions are given by Harrison (i945). Needham (1936^). The whole set of five longitudinal veins does not, however, always behave as a unit. There are certain genes which have locaHsed effects on particular veins or particular parts of veins. House (1952) has been able to show that some genes which appear to have strictly localised effects may exert on neighbouring regions sub-threshold influences which are not strong enough to produce any actual alteration except in combmation with other genes; but even if this is the case, it remains true that these genes affect most strongly particular sections of the venation rather than the system as a whole. One gene of this kind which acts at an early stage is cubitus interruptus. This causes an absence of the distal end of L4 in the prepupal stage, and this vein does not reappear and is also missing m the adult. It is interesting to find that in the aduk wing the tip of L5 moves forwards, as though to try to fill the space left by the absence of L4. We have, then, a certain reaction of the pattern as a whole to the local defect which is the primary effect of the gene. This reaction of the whole system probably occurs at the second phase, that of the pupal contraction, the absence of L4 having been produced earher, in the prepupal phase. Most local absences of veins, such as those caused by veinlet, tilt or radius incompletus, do not occur until the second phase, and in these cases there INDIVIDUATION— FORMATION OF PATTERN AND SHAPE 427 IS no sign of any general reaction of the pattern to compensate for the local absence. The last-formed element in the pattern— the posterior cross-vein— always adjusts its position in relation to any previous event which has affected its general neighbourhood. For instance, if the longitudinal veins are splayed apart at their first inception, e.g. by dachs, or even by differ- ential growth immediately after their formation, e.g. in the mutant broad, then the posterior cross-vein moves inwards towards the base of the wing. It reacts to absences of the distal parts of the longitudinal veins L4 and Ls even when these are produced by genes acting as late as the pupal contraction, such as veinlet. It can also be moved by surgical opera- tions made at a similar time (Lees 1941). These variations in the position in which the posterior cross-vein appears make it very obvious that the pattern is an expression of an equilibrium resulting from the interaction of numerous factors. Two other observations on wing venation may be mentioned, to emphasise the complexity of the underlying epigenetic processes and the equilibrium character of the simple pattern which is normally produced. Timofeeff-Ressovsky (193 1) studied a gene Vti {venae transversae incom- pletae) in Drosophila funebris. This causes a break in the posterior cross- vein. He found it possible, by selection, to isolate different stocks, in one of which the break occurred at the anterior end of the vein, in another at the posterior end and in the third at both ends with equal frequency. The genetic differences between these stocks depended on numerous factors of small effect. Since each of the genes concerned is presumably producing its own specific effect, we must conclude that very many mdividual processes are involved in this very detailed determination of a small part of the venation pattern. Again, another gene in the same species, also studied by Timofeeff-Rissovsky (1934), produces an effect which can be considered as a general disturbance of the condition of equihbrium of the vein-forming processes; the effect is either that many parts of the vein are missing or that large amounts of extra vein material are formed. The thoroughness with which the venation pattern can be analysed, thanks to the large number of mutant forms available, has revealed a number of general points which are probably applicable to other cases of animal pattern about which we have as yet less actual knowledge, hi the first place we see that even a comparatively simple pattern, such as that of the Drosophila wing venation, may be composed of a number of different parts which arise relatively independently and in diis case at somewhat different times. Thus the main longitudinal veins L3, L4 and L5, pass 428 PRINCIPLES OF EMBRYOLOGY through two phases in the prepupal and pupal stages, while L2 and the posterior cross-vein belong to a different system, since they are not represented in the prepupal wing; and for the posterior cross-vein in particular we have clear evidence that it is determined at a later stage than the longitudinal veins. Each system within the pattern must have a com- plex epigenetic basis, since genes exist which can aker it in a number of different ways. Sometimes the system reacts as a whole, as in dachs (where the shape of the wing is important) or in shifted (where it is not) ; but different parts of the system may have their own characteristic properties, exhibited for instance in the localised effects of cubitus intermptus or tilt. If a part of the pattern is removed at an early stage in the developmental processes, compensatory phenomena may occur later. Finally we may note that when the initial mass of material is much larger than normal, as in blot, there is a tendency for the whole pattern to be dupHcated, the duphcate being a mirror image of the normal. All these facts force one to conclude that a pattern represents a gradually developing equiHbrium between a number of forces. The system of forces may be very complex, and include a large number of different items. This might suggest that it is almost hopeless to try to obtain any fuller understanding of the genesis of a pattern. However, although the total number of forces involved may be unmanageably large, it is quite probable that only a few of them play major roles. For mstance, the wing veins represent cavities wliich persist when the originally hollow sack of the wing contracts and presses out the body fluid with which it was originally fdled. It is clear that the tension in the wing epitheha, and the hydrostatic pressure of the body fluid, must be among the important factors with general effects on the process. T e other main element in the situation is the set of factors which determine that there shall be only four (and not perhaps five) longitudinal veins between the anterior and poster- ior margins of the wing. It might not seem too optimistic to hope that we could discover the nature of the processes which determine this major feature of the pattern. It must be admitted, however, that as yet we have scarcely any clue even as to the general type of phenomena which comes into question. Are we dealing with diffusion, with the elastic and viscous properties of the membranes, or with — ^what? (b) The pentadactyl limb As another example of an embryonic pattern we may briefly consider the appearance of the bony structures in the vertebrate limb, hi the normal hindlimb there is a single femur attached to the tibia and fibula, at the end of which are the five digits. This fundamental pattern first appears as INDIVIDUATION— FORMATION OF PATTERN AND SHAPE 429 condensations within the mass of loose mesenchyme which makes up the body of the limb-bud (Fig. 20.5). The first point we may notice is that the pattern is not at all closely dependent on the mass of material available. If a limb-bud is halved at an early stage, the complete pattern may appear within the half-sized part. There is indeed a considerable tendency in limb-buds which have been Figure 20.5 On the left, the structures in the right hindHmb-bud of a normal laj-day mouse embryo, projected on to the plane of the footplate. The mesenchy- mal thickenings for the femur {FE), fibula (FI), tibia {TI) and digits are visible. AER is the apical ectodermal ridge. On the right is the disturbed pattern found in a luxate homozygote. The pre-axial side of the limb-bud IS enlarged, but the blastema of the tibia is absent. (From Carter 1954.) disturbed in some way, for instance by transplantation to other sites, for a spontaneous subdivision to occur, so that dupHcate limbs are formed. These are nearly always mirror-images of one another. If the division takes place at a somewhat later stage and is incomplete, partial duphcation may occur, giving rise to structures with more than the normal number of toes. Such polydactylous limbs are also produced by a number of genetic factors, hi some of the extreme forms, particularly in birds (Reviewed: Waddington 1952^7, Landauer 1948) the genetically caused Polydactyly 430 PRINCIPLES OF EMBRYOLOGY may also be fundamentally a duplication. In other cases, however, the condition represents an alteration to the basic pattern by the addition of elements to it, rather than a dupHcation of a pattern which remains essentially unchanged. For instance, in the guinea-pig the forefoot nor- mally has four digits, and the hindfoot three. Wright (1935) has described a dominant gene which when heterozygous produces some tendency towards the formation of extra digits. By selection Wright built up a race in which the general genetic background was such that the hetero- zygote rather regularly had five toes on the forefeet and four on the hindfeet. There is no evidence that this represented a partial duplication of the normal pattern ; it seems rather to be a straightforward modification of it. Animals homozygous for the gene had feet with very large numbers of toes which again showed no evidence of representing multipHcations of the basic four- or three-toed patterns. It is noteworthy, as an indication of the complexity of the reactions whose equilibrium is represented by the normal pattern, that in order to obtain the regular addition of a single toe it was necessary not merely to have a single dose of the main gene, but also to select a large number of other appropriate genes in the genetic background. The development of low-grade polydactylous limbs in mammals has recently been carefully studied by Carter (1954) in the luxate strain of mice. Polydactyls occur both among the heterozygotes and the homo- zygotes for this gene (Fig. 20.6). The first effect noticeable during their development is an overgrowth of the anterior (pre-axial) side of the limb-bud. In this region the condensations of mesenchyme are irregular, and extra condensed regions may appear from which the supernumerary Figure 20.6 The hindfeet of two luxate heterozygote mice, ilKistrating various grades of Polydactyly. The left foot of the second animal (on the right of the draw- ing) is normal. (From Carter 1954.) INDIVIDUATION — FORMATION OF PATTERN AND SHAPE 43 I digits will be produced. It is a remarkable fact that in the homozygotes, which of course show the more extreme expressions of the gene, skeletal elements are frequently missing (not only pre-axial digits but also the tibia). Thus the same genetic influence, which at fairly low intensity produces polydactyly, in higher intensity has an effect of a rather opposite character. Carter tentatively suggests that a field responsible for producing the five-toed pattern, and perhaps inherent in the apical ectoderm (see p. 276) is in the luxate heterozygotes shifted anteriorly in relation to the underlying competent mesenchyme, Tliis draws into the process of limb formation material which would normally lie beyond the range of the limb-inducing influence, and tliis may account for the increased size of the pre-axial region. To explain the reduction in the limb skeleton in the extreme cases he supposes that the pattern-inducting field is shifted so far anteriorly that part of it overlies mesenchyme which is not competent to respond. This hypothesis provides a formal explanation of the facts, but still leaves one quite in the dark as to the nature of the processes which cause the limb mesenchyme to form a certain number of condensations. It is probably significant that in polydactylous limbs it is normally on the pre- axial side that the extra digits appear. Gabriel (1946) found that if the opposite (post-axial) side of the limb-bud of a polydactylous strain of chicks was inhibited in growth by treatment with colchicine the Poly- dactyly was exaggerated. This suggests that one aspect at least of the normal process of pattern formation involves a control of the pre-axial side by the post-axial, this control being weakened when the post-axial side is inhibited. It is perhaps simplest to imagine the control involving the diffusion of substances, but there is still no direct evidence of this. Indeed, once again one has to admit that we have no clear-cut indication even of the general category of process to which we ought to look to find an explanation of the apparently simple fact that an originally homo- geneous mass of mesenchyme begins to draw itself together into a num- ber of separate regions of condensation. Phenomena of this kind obviously lie at the basis of the whole of animal morphology and our almost total ignorance as to how they are brought about offers a challenge which it is to be hoped experimentalists will soon take up successfully. In the last few paragraphs we have considered the original initiation of the pattern of the limb skeleton. It must of course be remembered that this may undergo modifications of quite a fundamental character after its first formation. For instance, Tschumi (1953) showed that if the young limb-buds of the toad Xenopus are treated with colchicine there is a considerable inhibition of growth which leads to a reduction in the size 432 PRINCIPLES OF EMBRYOLOGY of the toes. The different toes do not react equally, and although they fall roughly into order from more sensitive to less sensitive, the effects are somewhat irregular as between different animals Tschumi foimd that if a toe were reduced below a certain minimal size at the time of its first appearance it did not persist in later development but eventually dis- appeared, probably owiug to a process of the nature of competitive inter- action, by which the other toes drew away from it all the available supplies of essential metabolites (cf. Lehmann 1953). Thus four-toed or three-toed conditions may arise secondarily, if there is a sufficient disparity in size in the digits as they are originally formed. One cannot close even such a short discussion of animal pattern as this without referring to the famous work of D'Arcy Thompson, On Growth and Form (19 16). By showing that many animal forms share certain mathe- matical properties with shapes that are known to arise in the inorganic world, D'Arcy Thompson had a most important influence, both in per- suading biologists that form offers a problem which should be analysed in causal terms, and in making it seem not too impossibly difficult for such an analysis to be carried out. These services were very great ones; but nevertheless nearly all this task of understanding remains for the future. A large part of D'Arcy Thompson's work dealt with one special category of forms, namely those of simple cells and of small groups of cells. Even if one accepted his discussion, which is framed in terms of the surface tension of liquid films — and would nowadays be rejected by many who feel that the cell membrane cannot be regarded as a liquid — still one would be forced to admit that the principles he discussed throw little light on the initiation of a pattern such as that of a pentadactyl limb. Again, he discussed the forms that arise from particular types of differ- ential growth, such as that which causes the shell of a gastropod or a cephalopod to be twisted into a spiral; but interesting though that is, it leaves unsolved the fundamental question of how the pattern of differ- ential growth rates arises in the first place. The most essential problem of form is one which cannot be approached by a mathematical analysis of the ways in which animal shapes become transformed during develop- ment. It is the question of how form originates from the formless, and demands either an experimental attack or a mathematical analysis of a different kind, perhaps similar to that begun by Turing. Perhaps the most cheering thing that can be said about the problem of form is that it does not, perhaps, pose itself in its full intensity as often as might appear at first sight. The majority of animal forms develop out of something which already has some degree of pattern within it. For instance, an egg, as we have frequently seen, is by no means the com- INDIVIDUATION— FORMATION OF PATTERN AND SHAPE 433 pletely uniform and homogeneous body which it seems, but has certain elements of structure which provide a basis on which the more elaborate patterns of later development may be constructed. Even in examples such as those we have chosen to discuss, of the Drosophila wing and the vertebrate limb-bud, where the earHest stages show no obvious sign of even a trace of inherent pattern, it may well be that this apparent homo- geneity is partly deceptive. Perhaps after all we are never confronted with the origin of pattern from the completely formless but only with increases of complexity of pattern. But even so, the problem of how this occurs is difficult enough and at present almost completely beyond our understanding. 4. Morphogenesis The word 'morphogenesis' is often used in a broad sense to refer to many aspects of development, but when used strictly it should mean the moulding of cells and tissues into definite shapes. Morphogenesis, like pattern formation, is thus a phenomenon which is involved in almost every instance of differentiation. It is clearly impossible to discuss every- thing that is known about it. We shall therefore consider only one or two examples which will serve to illustrate the nature, and the successes and limitations, of some of the approaches which have been made to the problem. [a) Movements of isolated cells One of the simplest types of morphogenetic process is oriented move- ment by isolated cells. Weiss (1933, 1945) has devoted particular atten-r tion to this as a factor in normal development. By experiments on cells growing in tissue culture, he has shown very clearly that when cells creep about by any form of amoeboid movement a very powerful influence on the direction of movement is exerted by the microstructure of the medium or surface on which the cells are placed. Thus if the cells are provided with a fibrous substratum, such as glass wool, they tend to creep along the fibres. The same is true when the fibres are sub-micro- scopic in scale. If a plasma clot is stretched, the protein micelles of which it is composed become partially oriented, and both the shape and the direction of movement of the cells fits into the ultra-structure of the medium (Fig. 20.7). Weiss points out that in a developing embryo the intercellular spaces are filled with a 'ground substance' or protein-containing jelly. The differ- ential growth of different parts will stretch this in certain ways, and by thus orientating the ultra-structure of the ground substance, influence 434 PRINCIPLES OF EMBRYOLOGY the direction of migration of any mobile cells present. Such processes almost certainly play a very important part in events such as the out- growth of nerve fibres and the migration of the neural crest cells in vertebrate embryos. They may also be involved in some of the morpho- genetic behaviour of rather more closely packed aggregations of cells. They are one of the mechanisms one might look to in attempting to understand the origin of the five-rayed pattern in the limb-bud, which was discussed above without reaching any conclusion as to the funda- mental mechanism involved. On the other hand, this mechanism can scarcely be held to account for the movements and behaviour of tissues such as those involved in the gastrulation of the Amphibia or the rolling up of the neural plate. Figure 20.7 Above, diagram of the orientation of a reticular matrix between two centres of contraction. Below, diagram of the effect of stretching (between the arrows) on the shape of mesenchymal cells growing in a reticular matrix. (After Weiss 1949.) Another investigation on the movement of isolated cells which may have a considerable bearing on morphogenetic processes in general is centred around the rather peculiar situation in the amoeboid slime moulds [Acrasiales). During one stage in their life-history these organisms exist as isolated amoeboid cells. If these are cultured on a surface of nutritive agar, they at first move about in an uncoordinated and haphazard way. Growth and cell division continues until the density has reached some INDIVIDUATION — FORMATION OF PATTERN AND SHAPE 435 critical value, when a new process starts. This is aggregation. The amoebae move together into streams which converge on a certain region, the aggregation centre, at which they become heaped together to form a sausage-shaped cell mass (Fig. 20.8). This mass then starts moving as a whole, creeping over the surface at a speed comparable to that of the individual amoebae (approximately 2 mm. per hour). After a time the FiGxjRE 20.8 Four stages in the aggregation o£ Dictyostelium from the amoeboid phase to a compact mass. (From Bonner 1952.) creeping movement stops, and the heap gradually rears itself up into a peg-like structure, with a lump of cells (which become spores) at the top of a thin stalk which stands on a small expanded base (Fig. 20.9). Now it has been shown (particularly by Bonner 1947, 1952) that the first phase of these morphogenetic movements, namely the aggregation, occurs under the influence of a substance, known as acrasin, which is given off by the amoebae. Each amoeba gives off this substance, and at the same time tends to move along any gradient of acrasin that may be present. If sufficient amoebae happen to close together they form a 436 PRINCIPLES OF EMBRYOLOGY centre of high acrasin production, and other amoebae will move towards them. The behaviour of the amoeboid stage of the slime mould is important in providing a clear-cut demonstration that cells can attract their like from a distance. It seems quite probable that processes of this kind play a part, along with the orientation of the ground substance, in controlling the migrations of isolated cells during embryonic development. One must remember, however, that even in the slime moulds the existence of external gradients of acrasin concentration is not sufficient to explain the whole range of the phenomena we observe. For instance, the movement of the sausage-shaped lumps of aggregated cells do not seem to be directly dependent on external acrasin gradients. In these movements, and in the processes leading to the formation of the peg-like fruiting body, Bonner Figure 20.9 Stages in the formation of a fruiting body from the aggregated mass of Dictyostelium cells. A is during the migration of the mass. In B and C the mass is settling down, in D and E the cells at the tip of the mass are becoming elongated stalk cells which push down the axis to the substratum and then raise the whole mass into a peg-like structure. (From Bonner 1952.) INDIVIDUATION — FORMATION OF PATTERN AND SHAPE 437 suggests that influences arising from the contact and adhesion between the walls of the individual cells must be involved in addition to the acrasin gradients. It is worth pointing out that the whole sequence of aggregation, move- ment of the aggregated mass, and eventual formation of the fruiting body, exhibits, as all organic development does, the formation of a definite pattern of differentiated organs as well as mere movement. Twitty (1949, Twitty and Niu 1954, Fhckinger 1952) has devoted considerable attention to the factors controlling the migration of the pigment-forming cells from the neural crest in Amphibia. He has shown that one of the main factors is a tendency for the cells to move away from each other. This tendency is increased when the cells are in a closely confined space, which makes it probable that the underlying cause is a movement away from concentrations of waste products. This is the re- verse of what we see in the slime moulds, in which in the aggregation phase the amoebae tend to move together. However, the pigment cells ^ in different species of newt exhibit rather different properties in this respect. Whereas those of Tritums rivularis merely tend to disperse and to remain dispersed, those of T. torosus, after first dispersing, then tend to move back together again into clumps. The location in which these clumps form is influenced by the underlying mesodermal structures, and again there are differences between the species in these mesodermal influences. For instance, some factor in the torosus mesoderm prevents the migration of the pigment cells beyond the dorsal margin of the yolk mass, but this impediment is not offered by the rivularis mesoderm. Its nature is still unknown. Twitty claims that there is little evidence that oriented fibrillar structures in the ground substance, such as those postulated by Weiss, play any part in controUing these pigment cell migrations. Abercrombie and Heayman (1952, 1953) have also studied the move- ments of isolated cells, in this case chick fibroblasts in tissue culture, and have emphasised the fact that contact between cells often brings their migration to a halt, these cells apparently having a strong tendency not to creep over one another. {b) Movements of tissues: amphibian gastrulation Probably the most fully studied instance of morphogenesis and pattern formation by a tissue is the gastrulation and development of the embry- onic axis in Amphibia. The morphogenetic aspect of this comprises the tissue movements of gastrulation, the rolling up of the neural plate into a neural groove and tube, and the subdivision of the sheet of mesoderm into a notochord with rows of somites on each side of it. The pattern formation 438 PRINCIPLES OF EMBRYOLOGY involves, firstly, the determination of the plane of bilateral symmetry of the egg, and then the appearance of regional differences within the organiser (head organisers, tail organisers, etc.) and of a dorso-lateral field within the mesoderm. Clearly the pattern formation and the morpho- genesis are inextricably involved with each other, and can only be separ- ated conceptually by roughly classifying some of the events as rather more chemical in nature (and therefore related to pattern formation) and others as more definitely physical (and therefore connected with morphogenesis). For convenience of discussion we shall start by considering some of the morphogenetic processes which various authors have postulated, and shall then consider the nature and development of the patterns (p. 455). During gastrulation we are confronted with massive streaming move- ments by which the tissues are moved from one place to another. One hypothesis about the causation of such movement would be to suppose that certain regions are undergoing more rapid growth than others and that the tissue streams are due to the expansion of the growing regions. However, careful studies, particularly by Pasteels (1942^, have shown that in gastrulating embryos mitosis goes on at a more or less uniform rate in all regions. Similarly Gillette (1944) has shown that differential mitosis cannot be held responsible for the roUing up of the neural plate into the neural tube in Amphibia. Thus differential growth, important though it may be at later stages, is certainly not a major factor in the morphogenesis of the gastrula-neurula stages. Again it might be suggested that a differential expansion of the cells in certain regions, caused by the imbibition of water, might be the underlying cause of the morphogenetic movements. It has been found, hov/ever, that there is very little change in specific gravity of the cells during neurulation, and there can thus be little inhibition of water (Brown, Hamburger and Schmidt, 1941), so this hypothesis also is in- adequate. Another type of process, which' could be postulated to account for changes in cell shape similar to those which might be produced by differ- ential absorption of water, is the formation of fibrous structures in the internal cytoplasm. One might expect that if the cytoplasm becomes fibrous in character, the fibres would tend to lie parallel to one another and give rise to an elongation of the cell, or parts of the cell. Such parallel orientation of fibres could, in favourable circumstances, be detected by polarised light, since it should cause some degree of double refraction m directions dependent on the sub-microscopic orientation. This undoubtedly occurs in the mitotic spindle; and membranes, such as the nuclear INDIVIDUATION — FORMATION OF PATTERN AND SHAPE 439 membrane and the external cytoplasmic membrane, often show a double refraction due to the orientation of the molecules composing them (Reviewed: Frey-WyssHng 1948). In most cells, however, there is httle evidence that the bulk of the internal cytoplasm also contains orientated structures of this kind, al- though during many of the early morphogenetic processes in amphibian embryos some cells assume elongated wedge-like or flask-like shapes, such as might be expected on this hypothesis. This occurs, for instance, in the cells lining the early blastopore, in the neural groove and in ectodermal in-foldings such as the lens. Polarised light cannot be used in these cases, since the cells are still full of yolk granules which are highly refractile and obscure the picture presented by the cytoplasm itself. However, the yolk granules are not spherical, but somewhat ovoid, and if the cytoplasm possessed any strongly oriented fibrillar structure, one might expect that the yolk granules would lie with their long axes parallel to it. In- spection of sectioned material does not reveal any clear evidence of such orientation (Waddington 1942c), and it is therefore probable that intra- cellular fibres play at best a very minor role in amphibian early morpho- genesis (at least, directly, cf. Lawrence et al. 1944). The nuclei, which are of course much larger than the yolk granules, are usually very defmitely orientated in the same direction as the main body of the cells, and the appearances strongly suggest that this is because they have been squeezed into these positions by the constraining cell walls, which would therefore appear to exert an important effect in determining the shape of the whole cell. Recent work on the forces producing morphogenetic change in the amphibian embryo has, in fact, been led from several points of view to attach great importance to the behaviour of cell membranes, both those between the cell and the external medium, and between cell and cell. As has been particularly emphasised by Holtfreter (1943 a, 1943-44), the external (cell-medium) membrane of the early amphibian egg has several peculiar properties. Its outer surface is more or less solid and non-adhesive, and it has a great capacity both for elastic expansion and contraction and for plastic flow. As the egg becomes segmented this surface layer keeps fusing up again across the newly appearing cleavage furrows, so that it remains as a continuous undivided sheet (the so-called 'coat') connecting the cleavage cells. It is, indeed, the main thing which holds the cells to- gether. If it is dissolved by treatment with alkaline solutions the cells fall apart, since there is little tendency for the internal cell membranes to adhere to one another at this stage. As well as binding the cells together, the coat has also important osmotic properties, being less permeable to 440 PRINCIPLES OF EMBRYOLOGY most substances than are the membranes bounding the inner surfaces of the cells (Fig. 20.10). There seems Httle doubt that expansions and contractions of the coat are important factors in early morphogenesis. Holtfreter pointed out that if unfertiHsed eggs are kept in media of different osmotic pressure for several days, the pigmented coat of the animal half tends to expand in a manner which reminds one of the expansion of the animal pole region of the blastula during gastrulation. Indeed, after treatment with Ringer solution, the expanded animal coat may dive into the interior of the egg in a way which quite closely simulates the process of invagination. This suggests that the coat has some inherent capacity to carry out such Figure 20.10 Fragment of a morula of the axolotl, showing the pigmented surface coat. (From Holtfreter I943-) movements, independently of the cellularisation of the underlying mater- ial. It would be interesting to know whether the expansion of the coat is connected with the formation of any oriented fibrous micro-structure within it. The fact that the expansion is greater in the one plane, in which the 'invagination' takes place, suggests that this is the case, but there has been no direct demonstration of it owing to the difficulty of polarised light studies on such material. In stages later than gastrulation the coat may also have important mor- phogenetic effects. If small groups of cell are placed in an air/hquid or hquid/hquid interface, the cell membranes will be disrupted if the surface tension is powerful enough. By using a number of interfaces of appro- priate surface tensions, Waddington (i942<:) showed that the effective strength of the coated surfaces of cells from gastrula and neurula stages gradually mcreases as development proceeds, and that as the neural plate INDIVIDUATION — FORMATION OF PATTERN AND SHAPE 44I rolls up into the neural groove the strength of the outer concave surface becomes markedly greater than that of the inner convex surface. Such an increase in the strength of the concave surface would be produced if the coat there w^as undergoing contraction and thickening, and it seems rather probable that this is occurring and is one of the major factors in causing the change of the neural plate into the neural groove (Fig. 20.11). The whole process of gastrulation cannot, however, be attributed to changes in the coat. Amphibian gastrulation involves two main types of movement for which an explanation has to be found. If one compares the shape of the presumptive areas in the late blastula with the configura- tions which they wiU have assumed at the end of gastrulation, the major changes would be accounted for if we suppose that an area lying in the 4(A 60- .«0»<^ Neural tissue, outer side e^"^ ,0 Neurol tissue, inner side y ^..•*'' Jo^ ** Epidermis **B y ,.0'' ^f^^ ^y^^' Mesoderm x.....--v^/-^ 50- ♦«C ^'^^.^^^ '^ T --' Endoderm .^ -^ G^ ^ y' ^ ^ ^ y' ^ ^ ^y' 40- -f Y^ YounQ Mid Eorly Neural Closed 90Strula Qostrulo neural plate groove neural tube FiGXJRE 20.11 The ukimate strength of various tissues in amphibian embryos. The figure for the blastocoel roof of the young gastrula (indicated by a cross) was found by pulling a small steel ball through the tissue with a magnet. The other figures were derived from observations of the rate at which the cell surfaces were disrupted when placed in the air-liquid interface of the three saponin solutions whose surface tensions are given at A, B and C. The measurements are approximate, but indicate the changes which occur, and the relative strengths of the surfaces. (From data of Waddington 1939ft, i942f.) 442 PRINCIPLES OF EMBRYOLOGY dorsal midline became longer from anterior to posterior and at the same time narrower from side to side, while in more lateral regions these two changes in dimensions "were less marked. Indeed, on the ventral side there would have to be some increase in the side-to-side dimension to compensate for the narrowing that occurs near the dorsal plane. This locally-variable change in dimensions constitutes the first factor to be accounted for. The second is the fact that the dorsal material, as it in- creases its anterior-posterior length, is tucked inside the blastocoele to form a primitive gut instead of merely protruding as a process sticking out from the region of the blastopore. Epidermis Head mesodbem Figure 20.12 A, B are drawings of two stages of exo-gastrulation in the axolotl; the ecto- derm lies above, and the endo-mesoderm, instead of moving inside it, is elongating towards the bottom of the picture. C is a diagrammatic section through a later stage. (After Holtfrcter 1933.) The second factor has attracted considerably more study than the first. It can fairly easily go wrong if embryos, particularly of the axolotl, are allowed to gastrulate in solutions of abnormal osmotic pressure. There is then often a failure of the mechanism by which the elongating dorsal material should be turned inwards, and part of the mesoderm and endo- derm (or even the whole of it) may move outwards from the blastopore, forming a so-called exogastrula. In less strongly affected cases it is the anterior mesoderm which fails to get inside, while the more posterior INDIVIDUATION — FORMATION OF PATTERN AND SHAPE 443 material does so. Holtfreter (1933) has described the process in detail. He showed that the regions of the neural system developed in such partial exogastrulae correspond very closely w^ith the extent of the mesoderm that is formed inside as opposed to outside, a fact w^hich provides a neat illustration of the developing pattern of anterior and posterior organisers, which we shall shortly discuss (p. 455) (Fig. 20.12). Figure 20.13 A, section through the future blastopore region of the axolotl, at a time when the invagination is indicated only by condensations of pigmented coat to which early flask cells are attached. B, semi-diagrammatic section through a gastrula, to show the flask cells lining the blastopore and archenteron. C, the endoderm cells forming the floor of the archenteron at a later stage ; cells which have broken contact with the surface have contracted their 'necks' into small pigmented lumps. (From Holtfreter 1943.) There are at least two factors involved in the in-turning mechanism. The most obvious is the formation of peculiarly shaped cells at the posi- tion at which the blastopore first appears. These are usually described as flask or bottle cells. The main cell body, in which the nucleus is situated, is roughly ovoid and is drawn out into a long, thin neck by which it is connected to the external surface in the blastopore region. The narrow- ness of the neck may be partly the result of the contraction of the coat over the site of the blastopore furrow, but it is probably also influenced by processes going on within the cell cytoplasm, since the neck region, from which yolk granules are absent, has been found to show double 444 PRINCIPLES OF EMBRYOLOGY refraction which would indicate the appearance of a fibrous structure of the cytoplasm (Waddington 1940). The neck is certainly very contractile, and its tendency to contract almost certainly plays a part in pulling the elongating dorsal material inwards and thus beginning the gastrulation process (Fig. 20.13). A movement of one piece of tissue towards the interior of another tissue mass can, however, occur with little sign of the participation of flask cells Figure 20.14 Engulfment by the endoderm. Row i, a small fragment of vmcoated endo- derm becomes incorporated into an endodermal substratum. Row 2, a fragment of blastoporal cells, partly covered by coat, behaves similarly but also forms a groove. Row 3, if the endodermal fragment is covered by ecto- derm, the latter at first spreads but then rounds up and becomes isolated. (From Holtfreter 1943-44.) in the procedure. Holtfreter (1943 -1944) has described how groups of blastoporal or endodermal cells, placed against a larger mass of endoderm, become as it were engulfed into it (Fig. 20.14). He has shown that im- portant factors in this situation are the adhesiveness between the mem- branes of different types of cells, and what he speaks of as the 'surface tensions' developed along such cell-to-cell or cell-to-medium interfaces. By placing in contact small groups of cells from different tissues, he was INDIVIDUATION — FORMATION OF PATTERN AND SHAPE 445 able to show very clearly that the adhesiveness of a cell for other cells of the same or different kinds may change considerably during the course of differentiation. Thus in some tissues the cells are very closely packed at certain stages, when their adhesiveness is high, but tend to become dis- sociated from each other at other times, as for instance when the neural crest cells break away and start to migrate separately between the ecto- derm and mesoderm. Again, combinations of different tissues sometimes show considerable mutual affinity, when they round up into a single mass, while at other stages the two tissues may tend to separate from one another (Holtfreter 1939). Such processes are undoubtedly very important in influencing the shapes of neighbouring masses of tissues in a developing embryo. Holtfreter has given a number of diagrams illustrating the effects of these mutual interactions on the forms assumed by tissues some way along in their differentiation (Fig. 20.15). At the stage of gastrulation, differentiation has not proceeded very far and one would not expect to fmd clear-cut differences in affinity, but FiGxiRE 20.15 Figure a, isolated ectoderm develops into an irregular epidermal tissue; b, ectoderm combined with some mesenchyme forms an epithelium ciliated on the outer surface ; c, ectoderm embedded in mesenchyme, inside another epidermal skin, forms a vesicle or tubule ciliated on the inner surface. (From Holtfreter 1939.) instead more gradual transitions between one region and another. Holt- freter gives some evidence that such graded differences exist. He is in- clined to interpret them as differences in surface tension. He points out that if one has two drops of different materials, A and B, in contact and both immersed in a medium C, and if the tension in the AC surface is greater than the sum of the tensions in the BC and BA surfaces, then the AC surface will contract, and the drop of ^ will in effect be engulfed by 446 PRINCIPLES OF EMBRYOLOGY the drop of B. This, Holtfreter suggests, may provide a model of the incorporation of groups of cells placed in contact with endoderm, and indeed of one of the factors involved in the normal invagination (Fig. 20.16). The general principle of the suggestion seems rather plausible, but some caution is advisable in using the term 'surface tension' in connection v^ith amphibian cells. Strictly speaking, a surface tension develops only in a liquid interface, and there seems httle doubt that the external membranes Figure 20.16 Surface tensions in two drops of different fluids A and B immersed jji a third C. of cells cannot be regarded as truly liquid, but must be supposed to possess a certain degree of rigidity or solidity. This does not make it impossible, however, for such surfaces to exhibit properties analogous to those of the tensions which would develop in truly liquid interfaces. Returning to the two drops A and B in contact with one another, the engulfment of ^ would also occur if there was a strong tendency for the area of contact between A and B to increase, and such a tendency would arise if the A and B membranes in some way attracted one another. Since the mem- branes are largely protein, they may be expected to exhibit an orderly disposition of chemically reactive groups, which will tend to become attached to the appropriate groups on some neighbouring surface if the two patterns fit, but not otherwise. Weiss (1947, 1949, 1950b) has emphasised the important part that may be played in development by the mobilisation at the surface of the cell of compounds that have a specific chemical reactivity which causes INDIVIDUATION — FORMATION OF PATTERN AND SHAPE 447 them to enter into combination with substances at the surface of other neighbouring cells. He has laid particular stress on the influence of such reactions in changing the constitution of the internal cytoplasm by anchor- ing certain constituents on the surface, and he suggests that this may provide a general mechanism of differentiation (Fig.19.3), It is not easy to admit the general importance of the hypothesis in connection with the differentiation of substance, if only because differentiation can proceed quite well in unicellular Protozoa, or in completely isolated amphibian notochord cells (Mookerjee 1953), or pigment-forming cells (Twitty and Niu 1954). But processes of this kind may play a major part in morpho- genesis. Schmitt (1940) has also discussed, in what one might call general- ised chemical terms, how cell surfaces might be bound together by a an intermediate layer which could react with both of them (Fig. 20.7), fl^-Vc' ««n H,o o s-^ & Histone Zippei action Figure 20.17 A represents a rather flat cuboidal cell; its surface where it makes contact with the next cell is highly solvated, and adhesion between the cells is slight. If a desolvating agent (calcium ions, or histone) is introduced, the cells will be drawn together as though by a zipp fastener, eventually (C) forming tall cells with considerably greater surface of mutual contact. (From Schmitt 1941.) 448 PRINCIPLES OF EMBRYOLOGY Whatever the detailed chemical mechanism, it seems legitimate to think of the membranes of cells which are in contact with one another as be- having as though they were being forced together, or forced apart, by a number of zipp-fasteners, each with a characteristic tension tending to extend the zipped region. Such a system would behave very like a group of cells with truly fluid membranes possessing true surface tensions. It is very probable that forces of a similar kind, arising from the adhesiveness of the cell membranes, play a part not only in the in-turning of the invagination stream but also in producing it. It is certain that during the elongation and narrowing of the dorsal material, and the compensating expansion of the more ventral parts of the gastrula, there are considerable changes in the mutual contacts between the cells. One evidence of this is the fact that the layer of tissue forming the blastula roof, which is several cells thick at the beginning of gastrulation, is re- duced to a thickness of little more than two cells by the time gastrulation Figure 20.18 Four successive stages in gastrulation in Tritums alpestris. A group of four, and a group of three, cells have been followed, to show the changes in arrangement of the cells accompanying the streaming towards the blasto- pore tip. (Original, from a time lapse cine film.) is completed. Again, if cells are watched approaching the blastopore lip it will be found that they have slid in between one another in such a fashion that a group originally arranged in Hnes lying parallel to the blastopore lip has become rearranged into a longer and narrower shape (Fig. 20.18). A similar interdigitating movement of cells continues on the floor of the neural groove. The elevation and folding together of the sides of the groove may, in fact, be partly caused by the continuous anterior-posterior stretching of its midline, just as the edges of a piece of rubber will roll up on each side of a hne of stretch. INDIVIDUATION — FORMATION OF PATTERN AND SHAPE 449 Anterior-posterior stretching continues also in the internal dorsal material, namely the presumptive notochord. One could explain the whole morphogenesis of the presumptive mesoderm, into a central notochord flanked by rows of somites, in terms of tendencies for cells to move together in particular regions (Fig. 20.19). We might suppose that, as the mesoderm develops, there is at first a very strong tendency for cells near the midline to increase their surfaces of mutual contact and thus to interdigitate and form a narrow coherent rod. If this adhesiveness fell off rapidly from the midline towards the sides, cells shghtly lateral to Figure 20.19 Diagram of cell arrangement in the mesoderm of the newt neurula, show- ing the developing notochord and somites. the midline might have such a strong tendency to move towards it that they were pulled out of contact with cells lying further away; and thus the notochord would be isolated as a long rod of tissue separated from the more lateral mesoderm by a gap. To explain the next stage, the formation of the somites, one would have to suppose that a tendency for cells to move closer together begins to appear in more lateral regions, being at first stronger in the anterior and falling off rapidly towards the posterior. Then a transverse split would appear behind the first group of aggregated cells, isolating the first pair of somites; and if the process gradually spread backwards, the whole series of somites would be successively formed. 450 PRINCIPLES OF EMBRYOLOGY The tendencies of cells to move together, which have been postulated in the above scheme, are in most cases real enough, as can be seen by examin- ing the embryos at various stages. The hypothetical element is the suggestion that these tendencies for particular types of aggregation are produced by forces arising from the adhesiveness of the cell membranes. Again, we know that such forces exist in isolated cells (cf Holtfreter 1947, 0 '1 1 j^rJi^sSH'jSk' ''""^i'pJ'V'^^. I^&^l Figure 20.20 Stages in the development of the rounded cells of the archenteron roof into the notochord in the newt. At stage b the cell boundaries are difficult to see; at c the chordal sheath begins to be formed. The whole process of develop- ment consists, in the main, in an increase in the relative extent of the cell surface. (From Mookerjee, Deuchar and Waddington 1953.) INDIVIDUATION — FORMATION OF PATTERN AND SHAPE 45I 19480). But are they operative among the cells of the gastrula? In most phases of gastrulation and neurulation we still have not enough detailed knowledge of the shapes of the cells adequately to judge the plausibiUty of this. One process has, however, been examined in some detail from this point of view, namely the formation of the notochord in the midHne of the invaginated mesoderm (Mookerjee, Deuchar and Waddington 1953)- h is quite clear here that the area of contact between cells does increase considerably as the notochord forms. In fact the cells of the noto- chord soon take up an arrangement which has been compared to a pile of coins, each cell being a more or less flat disc with the maximum possible contact with other similar cells. The later stages of differentiation of the notochord, in which the cells become vacuolated until they are mere dis- tended bags full of cell sap, might be regarded as a consequence of the transformation of more of the original cytoplasm into membrane, so as to increase still more the area of intercellular adhesion (Fig. 20.20). In this example the increase in cell contact is clearly one of the basic pheno- mena of morphogenesis, and it seems not unreasonable to accept it as a causal explanation of the events. Forces arising from the cell membranes may well be the prime cause of the changes in tissue configuration during the whole process of gastrulation and neurulation (Fig. 20.21). Figure 20.21 Semi-diagrammatic transverse sections of the neural plate of the newt. In A the edges of the plate are only just beginning to fold upwards; the central part of the plate is occupied by nearly equidimensional cells, while at the edge the cells have become columnar and greatly increased the area of cell- to-cell contact. B shows the centre of the plate at a rather later stage, when cells have all become elongated. {B from Lehmann 1945.) It must not be supposed, however, that cell-membrane forces are always the most important factors in causing morphogenetic change. Elaborate and definite shapes may indeed be assumed by single cells, as for instance in nematodes where the greater part of the excretory canal forms within the body of one cell. There is still one aspect of gastrulation movements which remains to 452 PRINCIPLES OF EMBRYOLOGY be discussed; that is the fact that they are strongly polarised. It was recognised by Spemann in the early experiments with organisers that if tissue from the blastopore region is grafted into another site in an embryo it tends to go on invaginating in its own original direction, although it very often becomes swung round into conformity with the gastrulation movements of the host. Particularly striking examples of the tendency of a graft to retain its own polarity and direction of invagination can be seen in anurans, such as Discoglossus, in which development is very rapid and the anterior-posterior elongation and lateral narrowing of the blasto- pore material particularly well marked (Waddington, 1941). It is still rather unclear whether this polarity is a property of pieces of tissue, i.e. of groups of cells in which, perhaps there is a gradient in the intensity of the cell-membrane forces, or whether it is inherent in the individual cells of which the tissue is composed. Hokfreter (1947) has given a somewhat diagrammatic drawing of the recently invaginated mesoderm, in which a polar structure of the individual cells is indicated, and he has also shown experimentally that isolated cells do develop a polarity of their own. However, if this cell polarity exists before gastrulation it is certainly by no means irrevocably fixed, since small parts of the presumptive meso- derm of the newt, cut out and replaced with the anterior-posterior axis reversed, may in some cases invaginate in perfect conformity with their surroundings and show no signs of reversed cellular polarity (cf p. 458). 5. Measurement of the forces and energy involved in morphogenesis During morphogenetic changes regions of tissue are shifted about bodily in space; that is to say, work is done, and energy must be expended. Many attempts have been made to divide the energy used by an embryo into a fraction required for simple maintenance of the living system, and another fraction devoted to the performance of this morphogenetic work. In practice it has been found extremely difficult to do this; the earlier work on the subject has been fully discussed by Needham (1931). It has indeed been difficult even to demonstrate the existence of a morphogenetic energy fraction, let alone to measure it. One of the most successful attempts to do so has been that of Tyler, summarised in his review of 1942. He compared the rates of development and the oxygen consumption of embryos derived from whole echinoderm eggs or from separated first blastomeres (half embryos) or from fused eggs (giant embryos). He found that the rate of respiration was nearly the same in all, but that half embryos took a longer, and giant ones a shorter, time to reach a given stage than did normals. Thus by the time they reach a given stage, the half embryos have consumed more and the giants less energy INDIVIDUATION — FORMATION OF PATTERN AND SHAPE 453 per unit mass than the normals. Tyler argues that this is a reflection of the varying amounts of morphogenetic work per unit mass which the different types have to perform. This may indeed well be so; but, as Needham (1942) points out, the slow-developing half embryos have not only got to do twice as much morphogenetic work per unit mass as the normals, but also have to maintain themselves for longer before reaching any particular stage; and it remains obscure how much of the extra energy goes to one purpose or to the other. If considerable quantities of energy were utiHsed for the performance of physical work, then there should be a measurable discrepancy between the decrease in the calorific value of an embryo as its yolk is consumed and the amount of heat which it gives out. The most thorough study of this question is that of Smith (1946), and no such difference was found. Various other authors have attempted to estimate the maximum fraction of the UtiHsed energy which can possibly be supposed to be devoted to such work. Tyler gives a figure of 30 per cent for echinoderm eggs. Tuft (1953) has reviewed the measurements on the rate of oxygen con- sumption which have been made on the developing eggs of a number of different species (insects, fish, Amphibia). He shows that the curves are often by no means simple, but may have a succession of phases of increas- ing, stationary or even decreasing rates. In the bug Rhodnius, the rate of oxygen consumption falls during a certain period. Tuft makes a calcula- tion, based on the supposition that the minimum of the rate indicates the maximum consumption which can be considered necessary for mainten- ance, and concludes that it is conceivable that as much as 15 per cent could be devoted to something else, such as morphogenetic work. The weak point in such an argument is of course the assumption that the maintenance requirements remain constant (Fig. 20.22). Little is as yet known about the biochemical systems by which energy is dehvered to the morphogenetic mechanism; probably they involve high-energy phosphate compounds (Barth and Barth 1951). The ease with which a process such as gastrulation is brought to a standstill (e.g. by thermal shocks, a wide range of chemical inhibitors, etc.) suggests that the process is a very sensitive one. The ultimate source of the energy for amphibian gastrulation is presumably caroohydrate, since, as we have seen (p. 203), the consumption of glycogen increases greatly in the blasto- pore region just when movement begins. It may well be, however, that other morphogenetic processes obtain their energy from other sources. Needham (193 1) claimed that it is a general rule that during embryonic life, the predominant source of energy is, in the earHest stages, carbohy- drate, then protein and fmally fat. More recent investigations (e.g. 454 PRINCIPLES OF EMBRYOLOGY Lovtrup 1953) show that, in Amphibia at least, the succession is really carbohydrate-fat-protein. Gregg and Ornstein (1953) found that certain of the morphogenetic processes studied by Holtfreter in explants (cf. p. 444) were more sensitive to one group of enzyme inhibitors, others to different ones, which suggests that they do not all derive their energy in the same way. 0-30 0-20- 0 10 - 2 4 RKodnius FiGXJRE 20.22 Changes in the rate of ox)''gen consumption during the development of single eggs of the bug Rhodnius, at three different temperatures {D.C., time of dorsal closure; scale at left and below). And the same for the anuran Xenopus(B.G., beginning of gastrulation, iV.F., neural fold stage; scale above and to right). (After Tuft 195 3.) A more direct approach to the problem of the energetics of morpho- genesis would be to measure the actual forces which are exerted. In one attempt to do this for amphibian gastrulation, a small steel ball was in- serted among the cells of the invaginating mesoderm, and a bar magnet placed nearby in such a position that the magnetic force on the ball tended to prevent its movement. From a series of such measurements, one can arrive at a rough figure for the maximum force which the gastrulation movements can exert. This was found to be about 0*34 dynes per mm.^ of the hemispherical surface of the ball (Waddington 1939/)). INDIVIDUATION — FORMATION OF PATTERN AND SHAPE 455 Selman (1955) has made similar measurements of the force of neuralisa- tion ; his method consisted in placing two minute steel dumbbells against the two neural ridges, and holding them apart by placing the whole preparation within a coil carrying an alternating current; he found a value of 40 X 10 ^ dynes. Using an estimate of the distance over which the tissues move, one can roughly estimate the total amount of work accom- plished: during gastrulation it is about 3 x lO"^ cal. per hour. Even if one supposed that the energy-producing mechanism was working at an efficiency of only 10 per cent, the amount of oxygen consumed during the period could produce about a milHon times as much energy as this, so that these figures suggest that morphogenetic work demands only a very minute fraction of the energy available. It should be emphasised, however, that the measurements of the forces are of a very prehminary character; they may be too small, because the cells have been damaged during the experiment, or alternatively they may be larger than the forces actually exerted, since the measurement is of the maximum the tissues can put out rather than what they normally do. A measurement of the force of gastrulation has been made by another method in echinoderms by Moore (1941, 1945). If an echinoderm egg is cultured from fertihsation onwards in sucrose solution, some of this be- comes enclosed in the blastocoel, but when this cavity is fully formed the solution cannot escape since the walls of the blastula are impermeable to the sugar, which therefore exerts an osmotic pressure, directed outwards, if the blastula is transferred to sea water. By finding what concentration of sucrose just prevents the in-pushing of the endoderm, Moore decided that the force of gastrulation in this form is about 5 gm. per mm.^. This is about 10* as great as that found for the very different amphibian gastru- lation process. Even so, as Moore points out, the work done in echinoderm gastrulation would only demand about one thousandth of the oxygen which is actually consumed. 6. Individuation of the central nervous system in Amphibia The development of the nervous system from the gastrula to the neurula stage in Amphibia provides probably the best example from which one can get an idea of what is actually involved in the individuation of most embryonic organs. Not only has the process been very thoroughly studied in a long series of experiments by many authors, but it involves simultan- eously both the aspects of individuation — morphogenesis and pattern formation — which we have just considered separately. In reahty they must usually occur in combination with one another, and the develop- ment of the nervous system therefore gives a more generally valid picture 456 PRINCIPLES OF EMBRYOLOGY than the rather special cases we have chosen to exhibit the details of each process separately. Moreover, in the individuation of the nervous system a process of induction is very clearly involved and the pattern of the nervous system itself is partly derived from that of the underlying mesoderm. This is perhaps rather a special feature v^hich is not found, at any rate with such clarity, in the development of many other organs, but it has the advantage that it offers opportunities for experimental analysis which would not otherwise be possible. By the time the blastopore has become reduced to a narrow slit, gastru- lation may be said to be complete (although in the immediate neighbour- hood of the blastopore invagination of mesoderm will continue for some time longer). The structure of the sheet of mesoderm which acts as the inducer of the neural plate is at this time as follows. At its most anterior end it is thin and stretches widely from side to side; this part lies in front of the future notochord and is known as the prechordal plate. Posterior to it is the main mass of the chorda-mesoderm, the dorsal part of which will become notochord, the lateral part somites, v^th the intermediate meso- derm and the side-plate mesoderm still more laterally. Overlying the whole mesoderm is the ectoderm on which a neural plate is, or shortly will be, dehmited by the appearance of the neural ridges. From anterior to posterior the neural plate can be divided into four main regions. The most anterior of these, wliich overlies the prechordal plate, will become the forebrain (which is also commonly known as prosencephalon, but by some authors (e.g., Lehmann) as the archencephalon, and by others (e.g., Dalcq) as the acrencephalon). This develops into the two vesicles of the forebrain, the second of which bears the eyes, and it becomes associated with the nasal placodes. Posterior to it is the region which will become the midbrain and hindbrain which are collectively known as the deuter- encephalon and are associated with the ears. The anterior tip of the chorda lies somewhere within tliis region. Further posteriorly is the spinal or trunk region ; and finally the most posterior end of the neural plate con- sists of material which is not truly neural at all but will form part of the mesoderm of the tail. The neural plate also has a certain structure in the transverse plane, hi the dorsal midline the tissue is rather thin, forming a shallow groove. On each side of this lies the main bulk of the neural plate, wliich will form the walls of the neural tube. At the two edges are the neural ridges which will develop into the neural crest and even- tually form pigment cells, parts of the spinal nerves and certain ectomeso- dermal derivatives, such as some of the cartilages of the head. The question immediately arises how the pattern of regions arising within the neural plate is related to patterns which may be present in the INDIVIDUATION— FORMATION OF PATTERN AND SHAPE 457 inducing mesoderm. Spemann (193 1) pointed out that the inductive capacities of the difFerent regions of the mesoderm were not all the same even at the beginning of gastrulation. The presumptive anterior regions tend, other things being equal, to induce more anterior parts of the neural plate than do presumptive posterior regions. Shortly after this. Mangold (1933) showed that if different regions from anterior to posterior are cut out from the archenteron roof and implanted into young gastrulae they show characteristic differences in the region of the nervous system which they induce. It seemed then that one could consider the mesoderm from the early gastrula stage onwards as having a fairly high degree of regional specificity, so that the presumptive anterior part could be con- sidered as a 'head organiser' and the presumptive posterior part as a 'trunk or tail organiser'. In agreement with this Hall (1937) showed that if the blastopore lip is removed from a young gastrula and replaced by the lip from a consider- ably older gastrula with a yolk plug, this presumptively posterior graft failed to induce a head, the neural system of the resulting embryo having a spinal character right to its anterior tip. Holtfreter (1936) has given a map of these head organisers and trunk organisers (Fig. 10.3, p. 178). Later authors have claimed that there are more than two regions— the head and trunk— which behave independently of one another. Lehmann (1945) and Dalcq (1947) have both argued that one must consider at least the three main regions mentioned above, namely the forebrain, the mid-brain, and hindbrain and the trunk regions. Nieuwkoop (1947) presents evidence that in secondary induced embryonic axes the brain is always fully formed up to a certain level, anterior to which it is altogether absent, and on this basis he distinguished at least seven successive in- dependent zones. The experimental results always made it clear that the regional speci- ficity of an organiser was not something absolutely fixed in the sense that the organiser could induce only one specific region of the neural plate and nothing else. The results indicated at most a tendency for the in- duction to have a certain regional character, but trunk organisers, for instance, could sometimes induce more anterior parts and vice versa. These disturbances in the simple picture can be to some extent accounted for, at least in experiments in which the organisers are grafted into whole embryos, by the supposition that the region of the host embryo in which the graft lies exerts an influence on the regional character of the material induced. However, a certain degree of latitude iu the specificity of a particular region of the mesoderm is found even when it induces in isolated pieces of ectoderm removed from the influence of any host 458 PRINCIPLES OF EMBRYOLOGY embryo. (Extensive studies on regional specificity during gastrula stages have been made by Okada and a group of Japanese workers (see Hama 1950), but seem to suffer from lack of adequate statistical evaluation.) One is forced to admit that the regional character of the various parts of the mesoderm is only imprecisely determined during the process of gastrulation and is still to some extent labile. This is indeed what one might expect from other types of experimental evidence. Thus, as pointed out earUer (p. 190) Yamada (1940) has shov^oi that even in the neurula the 'level' of the mesoderm on the dorso-ventral axis is not yet fmally fixed; for instance, presumptive lateral plate can be forced to develop into somitic muscle if notochord is brought into its neighbourhood. A similar flexibility occurs along the anterior-posterior axis. Waddington and Yao (1950) showed that if presumptive anterior or posterior portions of the organiser are exchanged in young newt gastrulae, completely normal individuals may be produced, which must involve an alteration in the anterior-posterior specificity of the exchanged region. (In similar experiments with the rapidly developing gastrula of the anuran Discoglossus [Waddington 1941] the morphogenetic tendencies of a graft were so strong that they prevented its incorporation by the host and no redetermination of the regional character could be proved in that case.) Even at the end of gastrulation considerable flexibility still persists along the anterior-posterior axis, since a fairly normal embryo (with over-thick mesoderm) may develop if an extra archenteron roof is added with reversed orientation between the normal archenteron roof and the presumptive neural plate (Fig. 20.23). Figure 20.23 Some experiments on the regional properties of the organiser. On the left, the presumptive neural plate of a late gastrula folded back, a second archen- teron roof laid with reversed orientation over the original roof, and the ectoderm returned to place. On the right, reversal of the dorsal lip region. (From Waddington and Yao 1950.) INDIVIDUATION — FORMATION OF PATTERN AND SHAPE 459 Even at the neural plate stage, the regional specificity is not completely fixed, either in the neural tissue or in the mesoderm underlying it. This was clearly shown in extensive experiments by ter Horst (1948). She separated the neural plate firom the archenteron roof, cut each into five transverse strips, and cultured these after wrapping them in pieces of young gastrula ectoderm, and observed both the differentiation of the isolate and the character of the induction it produced. The neural plate fifths on the whole developed into their presumptive fate, but showed a tendency to produce the next most anterior and most posterior regions as well — thus they still have a capacity to regulate towards the formation of a more complete neural system. The differentiation of the mesoderm showed evidence of another kind for a lack of full determination — the anterior parts of it sometimes developed some neural cells. In their induction effects, the two tissues gave even more evidence of flexibility, each fifth inducing a certain region in greatest frequency, but also calling forth neighbouring regions in considerable numbers (Fig. 20.24). A re- markable fact is that a given fifth of the archenteron roof tends to induce Figure 20.24 The open neural plate of the newt is divided into five equal zones, from anterior (1/5) to posterior (5/5); the neural plate is dissected free from the archenteron roof; and neural plate and mesoderm are then separately com- bined with flaps of young gastrula ectoderm. The thick lines show the frequency of inductions of fore-, mid-, and hind-brains by the neural plate, and the thin lines that by the mesoderm. Note that the neural plate has a more 'anterior' performance than the corresponding mesoderm. (After ter Horst 1948.) 460 PRINCIPLES OF EMBRYOLOGY more caudal regions than does the overlying part of the neural plate. This may perhaps find its explanation in Nieuwkoop's hypothesis of a second phase of the induction process, in which the induced material is gradually transformed into more posterior regions of the axis (p. 462). We find, therefore, that during the process of gastrulation, while the mesoderm is exerting its inducing influence, it is itself only just in the process of acquiring a pattern of regional specificities. It is these half- formed specificities which must be responsible for the regionally different types of induction which the various areas of the mesoderm exert. The main question which has been debated recently is whether the specificities within the mesoderm, and the various types of induction for which they are responsible, are to be explained in terms of quantitative variations in the concentration of some one substance, or whether the facts require us to consider that different substances are being produced, characteristic of the different regions. One may probably assume that by the time histological differences can be recognised within the mesoderm sheet the various regions have come to be characterised by particular substances. We have seen (p. 216) that investigations on the inductive capacities of different adult tissues have led to the conclusion that there are at least two different inducing substances, one for forebrain and another for trunk-tail (or per- haps solely for mesoderm). The question is, have such chemical differ- ences arisen already at the time of gastrulation when the induction of the neural plate first occurs, or is the individuation of that original induction dependent only on a pattern of quantitative differences in the mesoderm ? It does not seem possible at present to give a perfectly firm answer to this question. Most authors, however, seem to be agreed that the meso- derm behaves as though composed of at least two different systems, one corresponding to the prechordal plate and the other to the main mass of chorda-mesoderm. It is to be presumed that these are chemically distinct, but there is some divergence of opinion as to exactly what effects the two regions produce, and as to the importance of quantitative variations within them. Dalcq (1947) has recorded a series of investigations in which the young gastrulae of Discoglossus were cut into two portions along some parallel of latitude which separated the organisation centre into a more animal (presumptively posterior) portion and a more vegetative pre- sumptively anterior portion. The animal part is then rotated through 180 degrees and replaced on the lower in such a way that the posterior organiser it contained lay on the ventral side (Fig. 20.25). Both parts of the organiser invaginate and induce partial embryos. From the study of such incomplete embryos, Dalcq came to the conclusion that the pre- chordal plate always induces the forebrain or associated structures, and INDIVIDUATION — FORMATION OF PATTERN AND SHAPE 46I that its effects can be arranged in a series of grades corresponding to the amount of inducing material present. The lowest grade of induction, he claims, is an isolated pineal body. When larger quantities of prechordal plate are present a small forebrain vesicle is induced. The next stage in- duces the appearance of a fmger-like anterior expansion which is capable of inducing an olfactory placode. With still more intense inductive action the eyes appear. Dalcq suggests that similar variations in effectiveness, dependent on quantitative differences in the inducer, occur also in more posterior parts Figure 20.25 Figure a, a young gastrula of the anuran Discoglossus is marked with a vital stain above the blastopore, and then cut in two along a parallel of latitude; b, the upper half is rotated through 180 degrees and replaced; c, a normal embryo is usually formed on the original dorsal side (unless the cut was too low), and a partial embryo, lacking some part of the anterior region, appears on the ventral side. (After Dalcq 1947.) of the nervous system overlying the chordamesoderm. The most clear-cut evidence in regard to these regions concerns the medio-lateral extent of the plate rather than its anterior-posterior structure. There is considerable evidence that the inductive capacity of the chorda-mesoderm is most powerful in the midhne and decreases on either side. Thus it can be shown that at the late gastrula stage the lateral plate mesoderm has a weak capacity of induction which can be effective on the highly reactive ecto- derm of the young gastrula stage, but not on the less-reactive ectoderm of older gastrulae (Waddington 1936^). Raven and Kloos (1945) have made grafts from medial or lateral parts of the archenteron roof underlying the neural plate and showed that the lateral ones were weaker inducers. Again, two of Dalcq's students, Damas (1947) and Gallera (1947), have studied the effects of cutting short the period in which induction can proceed. At 462 PRINCIPLES OF EMBRYOLOGY various times after the presumptive neural plate had been underlain by mesoderm, they removed small fragments of the plate, cleared them of adhering mesoderm cells and transplanted them to the ventral side of another embryo. They found that ectoderm which had been acted on for only a short time tended to differentiate into neural crest rather than into neural material proper. There seems little doubt then that neural crest presents a weaker grade of induction than neural tissue. These experiments do not, however, provide direct evidence that differences along the anterior-posterior axis (e.g. differences between midbrain and hindbrain, trunk, tail, etc.) also represent a series of grades of the strength of the inducing action. A somewhat different account of the action of the prechordal plate and the chorda-mesoderm has been given by Nieuwkoop (1952). He had the ingenious idea of joining elongated flaps of ectoderm to the gastrula in such a way that the base was near the archenteron roof, but there was quite a long extent of ectoderm into which the inducing material could diffuse. He found that in general the regional character of the structures induced at the base of the ectoderm flap was the same as that of the region to which it was joined, and that the induced structures extended from there towards the forebrain, which might be located at the free end of the process of ectoderm, although sometimes the inductions were in- complete and lacked the most anterior regions. Nieuwkoop interpreted his results by the hypothesis that all parts of the archenteron roof (chorda- mesoderm as well as prechordal plate) exert a first inducing action which stimulates the ectoderm to develop into forebrain-like organs; after this has occurred the chorda-mesoderm, if it reaches the ectoderm in question, exerts a second 'transforming' activity which changes the development of the induced material towards the production of some more posterior level of the axis (Fig. 20.26). Such a hypothesis fits in well with the ideas developed by Toivonen from his studies on the inducing powers of adult organs (p. 216). However, one cannot help feeling that the results described by Nieuwkoop scarcely prove his hypothesis. The phenomena in the ectoderm flaps, which take on the regional character of the point at which they are attached and exhibit the structures which would normally he anterior to this, rei^^ one very much of the appearances in a limb regenerate, the base of which develops the regionahty of the cut face on which it forms and which contains all the structures lying distal to this. In the case of the limb regenerate, it scarcely seems plausible to suggest that the first phase is the determination of the distal tip of the limb, and the second phase a trans- formation into more posterior regions; and such a hypothesis, though INDIVIDUATION — FORMATION OF PATTERN AND SHAPE 463 perhaps more plausible in Nieuwkoop's case, does not seem by any means necessitated by the data. His hypothesis is, however, supported by the recent work of Eyal- Giladi (1954), who claims to have shown that when any part of the meso- derm acts on ectoderm the first result is to confer on the latter the capacity to differentiate into neural crest, but that this stage is fairly rapidly passed through, and in the next period the ectoderm is always induced to form y.^ Figure 20.26 Flaps of competent gastrula ectoderm are grafted on to a host embryo in such a way as to become attached to the forebrain, hindbrain or spinal cord. The differentiation of the flaps is indicated by : dotted outline, epidermal ; cross-hatched, mesectodermal ; longitudinal lines, forebrain; crosses, more posterior neural regions. The diagram illustrates Nieuwkoop's interpreta- tion, which supposes that at first the whole of the induced region acquires a tendency to form forebrain, and that in the more posterior flaps this is later transformed towards more spinal development. (From Nieuwkoop 1952.) forebrain, while if the inductive action lasts even longer the transforming action will cause the production of more posterior structures. However, the stage when the ectoderm will develop only into forebrain was not noticed by Mangold and von Woellwarth (1950) who isolated pre- sumptive neural tissue from the mid-gastrula of Triton, and its existence can perhaps not yet be taken as certain. It will be clear from the last few paragraphs that most recent authors consider that at most very few different substances are involved in the regional determination of the neural plate. Dalcq discusses the question in terms of two, one for the prechordal plate and one for the rest of the chorda-mesoderm. Nieuwkoop considers that there are only two factors, 464 PRINCIPLES OF EMBRYOLOGY those responsible for the initial forebrain induction and the later trans- forming action. If the whole regional determination is to be attributed to quantitative variations in so few substances the responsibihty for most of the details of the structure of the neural system must be attributed to processes of self-individuation; that is to say, one must suppose that the process of induction gives to a particular region only a general specifica- tion to form forebrain or midbrain or some other part, and that the details of the structure of the organ are elaborated by processes going on within the reacting material. As Lehmann (i948/>) has pointed out, a self-individuating piece of tissue seems usually to produce a certain range of structures quite completely, and to lack the structures outside that range altogether, rather than to cause the appearance of something which could be considered as a re- duced edition of the whole. We have seen an example of this in Nieuw- koop's fmding (p. 457) that an induced embryo which contains the trunk will also contain all levels of the brain up to some point at which it stops, the more anterior levels being completely absent. This can be interpreted as indicating, not that there are separate different inducing substances for each level which may or may not be present, but that a given mass of induced neural tissue moulds itself into a neural system which is fully formed as far as it goes and misses out the other parts entirely (Fig. 20.27). The occurrence of self-individuation in the mesoderm as well as in the neural system has been very strikingly demonstrated by Holtfreter (1943 1)). He cut out the blastopore Up region from a young gastrula of Triton and treated it with alkahne solution. The tissue then becomes disaggregated and the cells fall apart. They can be thoroughly stirred round so that their original arrangement is quite lost (as can be demon- strated by mixing together cells from a vitally stained with those from an unstained embryo). The loose cells can then be caused to aggregate again by placing them in acid saline. After re-aggregation is complete the mass can be implanted into the blastocoele of the host embryo; and it is then found to differentiate into coherently arranged tissues and to induce a neural axis which has well-defined regional properties. Thus the re-aggregated organiser cells have produced within themselves a fairly high degree of morphological organisation, clearly by a process of self- individuation. We have so far considered the interaction between the sheet of inducing mesoderm and the overlying ectoderm as though these two remained the whole time in stationary contact with one another. It is, of course, clear that this is not really the case. The patterns of regional differences within the mesoderm and overlying neural plate arc coming into being at a time INDIVIDUATION — FORMATION OF PATTERN AND SHAPE 465 when energetic morphogenetic movements are going on; the mesoderm is invaginating at the blastopore and is moving forward under the pre- sumptive neural plate, which at the same time is streaming backwards above it towards the blastopore. Nothing very defmite is known about the significance of these movements for the processes of regionalisation, but it seems almost impossible to beheve that they are in fact without importance. It seems probable, indeed, that it is to the movements of gastrulation that one should look for an explanation of the field of quanti- tative variation in inducing capacity which all authors seem to agree must exist witliin the mesoderm. If an evocating substance begins to be re- leased in the mesoderm at the time this invaginates through the blastopore, Figure 20.27 Diagram to illustrate the results of partial inhibition of the development of the brain. The upper row shows, on the left, the normal situation with two fields, for forebrain and for mid- and hindbrain. On the right of this row are three stages of inhibition ; note that the more posterior field is not reduced at all until the anterior one has disappeared entirely. Below are diagrams of the types of brain corresponding to the fields, with indications of the tele- cephalon, diencephalon, mesencephalon and myelencephalon. (From Leh- mann 1948.) 466 PRINCIPLES OF EMBRYOLOGY by the late gastrula stage it would presumably have attained a higher con- centration in the more anterior mesoderm, which has been invaginated for a longer time, than in the more posterior regions. Similarly one might expect it to spread out laterally from the dorsal midline. Processes of this kind will give rise to a graded field of evocator concentration within the mesoderm (Waddington 1940^, Waddington and Yao 1950) (Fig. 20.28). lit 1 1 1 > I V ^ /III /^ / I > 1 1 f > ' I V V V ^^^_- Figure 20.28 To illustrate the time relations of organiser action. The drawing on the left represents the archenteron roof in a late gastrula, the black circle being the blastopore. If we suppose that the evocator is liberated in the median strip (close dots), and diffuses laterally, it will have spread into a field represented by the dashed contours. The drawing on the right represents the pre- sumptive neural plate, which has been moving down towards the blasto- pore, passing over the already invaginated mesoderm. The concentration of evocator which has diffused into it from below will be approximately as indicated by the contours. (From Waddington and Yao 1950.) As these authors have also pointed out, the movement of invagination will have another consequence in that at any given time the posterior end of the neural plate will have been underlain by mesoderm for a longer period than the anterior end. It may be surmised that the period of time for which the inducing mesoderm acts may have an influence on the regional character of the neural tissue which is produced. Waddington and Deuchar (1952) attempted to demonstrate this. From a late gastrula stage, pieces of presumptive neural plate were removed and transplanted into young gastrula hosts in such a way that they were exposed to the INDIVIDUATION — FORMATION OF PATTERN AND SHAPE 467 action of the archenteron roof for a second time. It was hoped that by prolonging the period of induction in this way some change would be made in the regional character of the induced neural tissue, or even that it would be converted into posterior mesoderm which, in normal develop- ment, is the fate of that part of the neural plate which is longest underlain. Little effect was, however, produced. Nevertheless it remains true that during the induction of the neural plate we are dealing vdth a system which involves a considerable amount of relative movement, and it seems most probable that the time-relations both in the production of the evocating substance and in the period of its action on the overlying ecto- derm will eventually be found to play some part in the process. This summary of recent work on the induction of the neural plate, incomplete though it is, has probably sufficed to show how complex a problem is presented by even a comparatively simple instance of individua- tion. The more one looks into the details of what actually occurs in neural induction the more paradoxical the phenomena appear to be. For instance, we have seen that the neural crest appears to represent a weak grade of induction, yet the first visible sign of the formation of the neural plate occurs not in the dorsal midline but at the margins from which the neural crest will later develop; it is in this region that the cells first assume the elongated columnar shape which will later be characteristic of the whole neural epithelium (cf. Fig. 20.21, p. 451). Why should it be in a region of apparently weak action that an effect is first produced? Again, is it not paradoxical that it is at the anterior end of the neural plate, which is underlain for the shortest time by the archenteron roof, that the neural tube attains its greatest dimensions, while in more posterior regions it is smaller in cross-sectional area, and the very posterior end of the plate, which has been successively underlain by all the levels of the archenteron from anterior to posterior, does not develop into neural tissue at all but forms the mesoderm of the tail? We can certainly not pretend to have any explanation of these facts as yet. In spite of all the work that has been done on the regional determination of the neural plate, we still fmd ourselves forced to appeal to the myster- ious process of 'self-individuation' to explain the appearance of pattern. This is true both of the pattern in which the different inducing capacities are arranged in the mesoderm sheet, and of the details of the form of the neural organs such as the forebrain, midbrain, etc. By 'self-individuation we in fact mean no more than that the pattern arises without any ascertain- able antecedents. We have seen earlier that both Henke and Turing have discussed ways in which such spontaneous appearance of pattern might be supposed to occur; but it is unsatisfactory to have to rely on mechanisms 468 - PRINCIPLES OF EMBRYOLOGY of the kind they suggest to account for such complex forms as those of the parts of the brain, and we must certainly continue to search for more definite bases on which the patterns could be built. SUGGESTED READING Bonner 1952, Holtfreter 1943-44, Needham 1936a, Weiss 1950Z), Stem 1954. For an early and still most stimulating account of cell-specific adhesiveness (in sponges) see Huxley 1911, 1921. ACKNOWLEDGEMENTS It is a pleasure to express my gratitude to the numerous persons who have given me permission to utihse already-published figures. These include authors, editors of journals and publishers. The caption to each figure includes a reference to the work from which it was taken, the full title of which will be found in the Bibliography. Most of the original figures were drawn by Mr. E. D. Roberts, for whose care and skill I am most grateful. And to make an end is to make a beginning. The end is where we start from. •k -k -k -k -k We shall not cease from exploration And the end of all our exploring Will he to arrive where we started And know the place for the first time. From Little Gidding by T. S. EHot. BIBLIOGRAPHY The bibliographic reference has been given immediately after the author's name, in the form: Journal, volume number, number of first page. 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I95i 342 shape transformations, 290s shell gland, 104 silkw^orm, 139 skull, 234, 296 slime moulds, 434 somatoblasts, 96 somites, 8, 113, 258, 262, 449 sperm, 29s aster, 50 supernumerary, 62, 136 spiders, 36, 123 spindle, 51, 53, 55, 66, 70, 94, 99, 438 spiral symmetry, 43, 60, 66, 94s, 432 splanchnocoel, 263 Stentor, 395 steroids, 195, 212, 413 sturgeons, 59, 75, 225 subgerminal cavity, 155 submandibular gland 269 sulphydryl, 33. lOi. 205, 260, 355 surface tension, 72s, 416, 432, 440, 443, 445s Tachydnes, 128s tail-bud, 113, 167, 251, 263 tail fold, 251 teeth, 221 teleosts, 36, 226, 244s, 247s telolecithal, 36 temperature shocks, 212 Tenehrio, 128 tissue culture, 14, 160, 184, 237, 289, 361, 433s, 437 specificity, 215, 307, 400 tracers, 58, 91, 201, 204, 205, 206, 214, 384 trachea, 257 transformation, bacterial, 376, 400 transforming induction 462 trochophore, 94s trophoblast, 236 Tubifex, 42, 68, 74, 96, 98, loi, 119, 131 tumours, 307, 399 twins, identical, 286 insects, 123 mammals, 242, 248, 286 mosaic eggs, 60, 99 510 ultra sonics, 134 violet, 51, 65, 125, 140, 213, 382 umbilical cord, 262 union of nuclei, 50 Urechis, 45, 53, 68 vertebrae, 262 virus, 372, 375, 388, 389s, 399 vital staining, 93, loi, 158s, 224, 226s vitelline membrance, 38, 42, 146 SUBJECT INDEX vrool, 259 x-rays, 51, 141, 305, 306, 307, 333, 372, 373 Y chromosomes, 36 yeast, 401 yolk, 5, 36, 58, 201 gel membrane, 226, 229 nucleus, 34, 36 plug, 162 Wolffian duct, 266, 268 zip fastener, 446, 449