Waddington1956 20

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

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

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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 implied 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 midline, 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 configuration; or again, a neural plate rolls up into a neural tube, which is characterised by the well-defined swellings of the brain vesicles, etc. All 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

415 416 PRINCIPLES OF EMBRYOLOGY

sharp, but it is a useful rough classification and we shall see that in primary morphogenesis there are many factors which play a more important part than differences in growth rate.)

The two aspects of individuation—morphogenesis and pattern formation—are obviously closely connected with one another. It is hardly to be supposed that any complicated three-dimensional structure will develop unless the material out of which it is made has already developed a pattern of different properties in its various parts. Thus some degree of pattern formation probably always precedes any but the very simplest morphogenetic processes. Contrariwise it is to be expected that a developing pattern will be influenced by the shape of the area or mass in which it is forming and we shall find examples which demonstrate that this is the case. It is, however, helpful to use the distinction between pattern formation and morphogenesis as a means of arranging the subjects which require discussion into some sort of order. Moreover there is a certain difference in the kind of processes which must be involved in 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 involve processes which are both extended throughout a region of space and which also have a certain unity. As was suggested earlier (p. 23) such fields arise from the interaction of a number of different factors cach of which is extended throughout the region involved. We cannot expect, therefore, to be able to attribute the formation of a pattern to the action of any one single factor, but must expect always to find that several different things are involved in it, and the same expectation of a multiplicity of causes rather than a single cause holds goods for morphogenesis.

Although pattern formation and morphogenesis occur in the differentiation 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 ate very different in different cases. The shape of a mass of tissue, for instance, may in one case be altered by changes in the tension in the surface of the mass, or again by changes in 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 movements 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 find, 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.

1. 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 problem. 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 threshold 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 attainment 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 relation between the time taken for the hormone level to fall again by excretion 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 concentration 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 418 PRINCIPLES OF EMBRYOLOGY

according to the quantity of hormone injected and the rate of its excretion, quite different visible patterns might result; but they are all secondary consequences of the thresholds and time-lags which vary in a definite manner within the feather. It is the arrangement of these variations which must be regarded as the primary pattern. It is comparatively easy to understand how, once the primary pattern is given, various manifestations 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 Lepidoptera (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 ficld, 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 independent. 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. Morcover, 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 fully determined at a time at which others are still capable of being 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 Peripheral ° a“ eo ae field 4° 7 Central 7 '

FIGURE 20.1

On left: generalised scheme for the three main elements in the wing pattern in butterflies. Gf indicates the maximum extent of the General Field, Pf of the Peripheral Field (—=‘Umfeld’), and C uf 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 ordinate 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 determined. The three wings drawn on the graph illustrate the results of an carly operation, at a time when only the spots of the edge (General Field) are determined, then a case in which the Peripheral Field is determined 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 independent areas rather than a single one; but this complexity is compensated for by the great variety of patterns which are available in different species and the ease with which they can be studied.

Henke has discussed the different types of pattern which might theoretically 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 homogeneous. 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 calls a ‘spatter’ pattern. The next class he distinguishes is that in 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 lines at approximately equal distances apart. These he speaks of as ‘simultaneous rhythms’, distinguishing them in this way from other rhythmic patterns which involve time, which will be mentioned later. The simultaneous rhythm may be on a small scale in relation to the whole area covered, in which

FIGURE 20.2

Types of pattern, according to Henke, illustrated in lepidopteran wings: a, spatter pattern; b, c, d, e, ‘simultaneous rhythms’ of increasing wavelength, leading to the case f when there is only a single element present; g, a single spot which has a transitional zone around it; h, a diffusion field; i, accumulation of diffusing material at the boundary; k, 1, m, are other examples of boundary accumulations in more complex patterns; #, a “centric rhythm’ resulting from a Liesegang-like phenomenon in a diffusion field; o, p, 4,

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. Alternatively, 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 iniply 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, presumably 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 thythmic pattern may have not a sharp boundary but one which grades gradually into the background; or there may be a reaction between the spot and the background in such a way as to outline 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 sometimes sufficient to suggest that a given thythmic 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 formation of the pattern and a gradual increase or decrease in wave-length. A final distinction of the two types can, however, only be reached experimentally.

In a number of cases, indeed, experiment has demonstrated that diffusion 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 points, 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

FIGURE 20.3

Diffusion as the process producing the Central Field in the wing of the mealworm Ephestia. The upper row show examples of inhibition of the diffusion by cauterisations performed at the time when it would normally be proceeding (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 Sy; c, a similar narrowing caused by high-temperature treatment during the diffusion; d, a widened field produced by the gene Syb; e, a similar effect caused by early hightemperature 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 characteristic of the two areas.

How far does this account of some fundamental types of pattern formation, 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 within 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 reactions in any way, then the straightforward situation would be the attainment of some sort of balanced equilibrium condition throughout the whole area. But such an equilibrium is only a statistical phenomenon; actually the system will be disturbed by slight 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 with 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 cylindrical 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 this does not seem very convincing. One of the most important characteristics of embryonic development is that the patterns which arise tend to be accommodated 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 definite chemical wave-length dependent on the unchanging values of rate constants, 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 dapplings 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 suggests 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 understood.

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.

EE 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 fully analysed. Studics employing essentially biological methods (e.g. investigation 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, consisting of five longitudinal veins running from base to tip, with two crossveins, an anterior one between L3 and L4 and a posterior between L4 and Ls. This pattern arises in a series of stages, of which we may distinguish three: (1) The prepupal stage, in which L2 is absent, L3 and L4 are united 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 L1) and there are no cross-veins. At the end of this stage the wing is inflated into 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 appearance. (3) In a slightly later phase, at the very end of the contraction, the two cross-veins appear (Fig. 15-1, p. 331) 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 generally acting genes are, however, of several different kinds. Perhaps the most striking, but the least illuminating with regard to pattern formation, are genes such as dumpy which, by affecting the pupal contraction, distort the pattern of veins 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 all the veins from L2 to Ls appear as though squeezed together, diverging at a lesser angle. This occurs without any noticeable change in the outline 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 20.4

Gene-controlled modifications of the pattern of wing venation in Drosophila. In a, b, c, d, e the mutant wing is drawn in a full line, superposed on a dotted drawing of a normal wing; a, in shifted-2 the longitudinal veins are pinched together; b, in broad, the wing blade is relatively broader than normal and the veins diverge at a greater angle; c, veinlet may remove a considerable part of the distal region of the veins, and the posterior cross-vein is moved to fit; d, cubitus interruptus removes the distal part of the fourth vein, and the fifth then shifts upwards; e, dachs produces a short square wing, in which the veins diverge at a greater angle than normal; f, the dachsous wing is large and the veins diverge at a large angle, notice also the crossveins; 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 alteration 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 determined; 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 conditions under which it comes into being. Genes are known, however, which cause increases in the wing mass earlier 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 blot, 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 Ls. 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 discussions are given by Harrison (1945), Needham (1936a).

The whole set of five longitudinal veins does not, however, always behave as a unit. There are certain genes which have localised 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 combination 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 in the adult. It is interesting to find that in the adult wing the tip of Ls 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 carlier, 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 clement 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 differential 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 operations 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 (1931) studied a gene Vti (venae transversae incompletae) in Drosophila funebris. This causes a break in the posterior crossvein. 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 individual 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 equilibrium 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. In 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 this case at somewhat different times. Thus the main longitudinal veins L3, L4 and Ls, 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 complex epigenetic basis, since genes exist which can alter 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 interruptus 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 duplicated, the duplicate being a mirror image of the normal.

All these facts force one to conclude that a pattern represents a gradually developing equilibrium 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 instance, the wing veins represent cavities which persist when the originally hollow sack of the wing contracts and presses out the body fluid with which it was originally filled. It is clear that the tension in the wing epithelia, 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 posterior 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. In 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 hindlimb-bud of a normal 12}-day mouse embryo, projected on to the plane of the footplate. The mesenchymal 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 Iuxate 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 duplicate 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 duplication may occur, giving rise to structures with more than the normal number of toes.

Such polydactylous limbs are also produced by a number of genctic factors. In some of the extreme forms, particularly in birds (Reviewed: Waddington 1952a, 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 duplication of a pattern which remains essentially unchanged. For instance, in the guinea-pig the forefoot normally 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 heterozygote 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 multiplications 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 homozygotes 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 Iuxate heterozygote mice, illustrating various grades of polydactyly. The left foot of the second animal (on the right of the drawing) is normal. (From Carter 1954.) INDIVIDUATION—FORMATION OF PATTERN AND SHAPE 431

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. This draws into the process of limb formation material which would normally lie beyond the range of the limb-inducing influence, and this 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 preaxial 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 polydactyly 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 homogeneous mass of mesenchyme begins to draw itself together into a number 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 found 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 disappeared, probably owing to a process of the nature of competitive interaction, 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 (1916). By showing that many animal forms share certain mathematical properties with shapes that are known to arise in the inorganic world, D’Arcy Thompson had a most important influence, both in persuading 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 differential 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 differential 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 development. It is the question of how form originates from the formless, and demands cither 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 comINDIVIDUATION—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 earliest stages show no obvious sign of even a trace of inherent pattern, it may well be that this apparent homogeneity 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 everything 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.

(2) Movements of isolated cells

One of the simplest types of morphogenetic process is oriented movement by isolated cells. Weiss (1933, 1945) has devoted particular attention 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 ifthe 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-microscopic 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 differential 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 outgrowth of nerve fibres and the migration of the neural crest cells in vertebrate embryos. They may also be involved in some of the morphogenetic 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 fundamental 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. |

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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

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Four stages in the aggregation of 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 amocbae. 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

artes PTT

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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, movement 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, Flickinger 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 reverse of what we sec 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 Triturus 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 controlling these pigment cell migrations.

Abercrombie and Heayman (1952, 1953) have also studied the movements 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 embryonic 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 morphogenesis are inextricably involved with cach other, and can only be separated 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 o

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 movements 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 (1 9426), 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 rolling 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, however, 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 inadequate.

Another type of process, which could be postulated to account for changes in cell shape similar to those which might be produced by differential 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 in directions dependent on the sub-microscopic orientation. This undoubtedly occurs in the mitotic spindle; and membranes, such as the nuclear INDI VIDUATION—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-Wyssling 1948).

In most cells, however, there is little evidence that the bulk of the internal cytoplasm also contains orientated structures of this kind, although 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. Inspection of sectioned material does not reveal any clear evidence of such orientation (Waddington 1942c), and it is therefore probable that intracellular fibres play at best a very minor role in amphibian early morphogenesis (at least, directly, cf. Lawrence et al. 1944). The nuclei, which are of course much larger than the yolk granules, are usually very definitely 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 (19434, 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 together. 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

FF 440 PRINCIPLES OF EMBRYOLOGY

most substances than are the membranes bounding the inner surfaces of the cells (Fig. 20.10).

There seems little doubt that expansions and contractions of the coat are important factors in early morphogenesis. Holtfreter pointed out that if unfertilised 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 1943.)

movements, independently of the cellularisation of the underlying material. 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 morphogenetic effects. If small groups of cell are placed in an air/liquid or liquid/liquid interface, the cell membranes will be disrupted if the surface tension is powerful enough. By using a number of interfaces of appropriate surface tensions, Waddington (1942c) showed that the effective strength of the coated surfaces of cells from gastrula and neurula stages gradually increases 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 was 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 configurations which they will have assumed at the end of gastrulation, the major changes would be accounted for if we suppose that an area lying in the

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FIGURE 20.11

The ultimate 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, Band C. The measurements are approximate, but indicate the changes which occur, and the relative strengths of the surfaces. (From data of Waddington 19396, 1942c.) 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 increases 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.

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FIGURE 20.12

A, Bare 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 Holtfreter 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 endoderm (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 with the extent of the mesoderm that is formed inside as opposed to outside, a fact which provides a neat illustration of the developing pattern of anterior and posterior organisers, ° which we shall shortly discuss (p. 455) (Fig. 20.12).

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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 position 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 narrowness 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 1, a small fragment of uncoated endoderm 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 ectoderm, 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 important factors in this situation are the adhesiveness between the membranes 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 dissociated from each other at other times, as for instance when the neural _ crest cells break away and start to migrate separately between the ectoderm 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 find clear-cut differences in affinity, but

FIGURE 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. Holtfreter gives some evidence that such graded differences exist. He is inclined 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 A 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 with amphibian cells. Strictly speaking, a surface tension develops only in a liquid interface, and there seems little doubt that the external membranes

FIGURE 20.16

Surface tensions in two drops of different fluids A and B immersed in 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 A 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 membranes 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 anchoring 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 morphogenesis. Schmitt (1940) has also discussed, in what one might call generalised chemical terms, how cell surfaces might be bound together by a an intermediate layer which could react with both of them (Fig. 20.7).

g < % x Z A Xx Zg | y Z y % +Ca** or Histone B c

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. Ifa 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 y mutual contact. (From Schmitt 194. 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 behaving 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 reduced to a thickness of little more than two cells by the time gastrulation

3

FIGuRE 20.18

Four successive stages in gastrulation in Triturus 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 blastopore 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 lines 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 line 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 slightly lateral to

FIGURE 20.19

Diagram of cell arrangement in the mesoderm of the newt neurula, showing 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 ageregated 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 examining 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,

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 ¢ the chordal sheath begins to be formed. The whole process of development 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 4S5I

1948a). 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 plausibility of this. One process has, however, been examined in some detail from this point of view, namely the formation of the notochord in the midline of the invaginated mesoderm (Mookerjee, Deuchar and Waddington 1953). It is quite clear here that the area of contact between cells does increase considerably as the notochord forms. In fact the cells of the notochord 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 distended 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 phenomena 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 blastopore 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. Holtfreter (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 mesoderm 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 (193 1).

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 utilised 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 utilised 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 consumption 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 increasing, stationary or even decreasing rates. In the bug Rhodnius, the rate of oxygen consumption falls during a certain period. Tuft makes a calculation, based on the supposition that the minimum of the rate indicates the maximum consumption which can be considered necessary for maintenance, 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 delivered 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 cardohydrate, since, as we have seen (p. 203), the consumption of glycogen increases greatly in the blastopore region just when movement begins. It may well be, however, that other morphogenetic processes obtain their energy from other sources. Necdham (1931) claimed that it is a general rule that during embryonic life, the predominant source of energy is, in the earliest stages, carbohydrate, then protein and finally 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.

———_— Xenopus 2 4 6 8 10 12 14 16 18 20 22 24 |

T T T T T T T T T T T T

Age in hours 4-08

Age in days

2 4 6 8 10 12 14 16 18 20 22 24 26 28 Rhodnius ————>

FIGURE 20.22

Changes in the rate of oxygen 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, N.F., neural fold stage; scale above and to right). (After Tuft 195 3.)

A more direct approach to the problem of the energetics of morphogenesis would be to measure the actual forces which are exerted. In one attempt to do this for amphibian gastrulation, a small steel ball was inserted 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 neuralisation; 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 accomplished: during gastrulation it is about 3 x 107° 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 million 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 preliminary 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 fertilisation onwards in sucrose solution, some of this becomes 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 gastrulation 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 simultaneously both the aspects of individuation—morphogenesis and pattern formation—which we have just considered separately. In reality they must usually occur in combination with one another, and the development of the nervous system therefore gives a more generally valid picture

GG 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 which 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, gastrulation may be said to be complete (although in the immediate neighbourhood 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 isthe main mass of the chorda-mesoderm, the dorsal part of which will become notochord, the lateral part somites, with the intermediate mesoderm 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, delimited 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, which 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 deuterencephalon and are associated with the ears. The anterior tip of the chorda lies somewhere within this region. Further posteriorly is the spinal or trunk region; and finally the most posterior end of the neural plate consists 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. In 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, which will form the walls of the neural tube. At the two edges are the neural ridges which will develop into the neural crest and eventually form pigment cells, parts of the spinal nerves and certain ectomesodermal 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 (1931) 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 considered 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 considerably 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 independent zones.

The experimental results always made it clear that the regional specificity 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 induction 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 in 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 earlicr (p. 190) Yamada (1940) has shown that even in the neurula the ‘level’ of the mesoderm on the dorso-ventral axis is not yet finally 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 from 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 remarkable fact is that a given fifth of the archenteron roof tends to induce

100

Fore brain ———— Mid brain

ee = Hind brain 804

25

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 combined 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 halfformed 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 perhaps solely for mesoderm). The question is, have such chemical differences 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 mesoderm 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 presumptively 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 prechordal plate always induces the forebrain or associated structures, and INDIVIDUATION—FORMATION OF PATTERN AND SHAPE 461

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 induces the appearance of a finger-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 midline 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 ectoderm of the young gastrula stage, but not on the less-reactive ectoderm of older gastrulae (Waddington 19362). Raven and Kloos (194 5) 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 incomplete and lacked the most anterior regions. Nieuwkoop interpreted his results by the hypothesis that all parts of the archenteron roof (chordamesoderm 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 studics on the inducing powers of adult organs (p. 216). However, one cannot help fecling that the results described by Niewwkoop 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 lie anterior to this, remind one very much of the appearances in a limb regenerate, the base of which develops the regionality 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 transformation 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 EyalGiladi (1954), who claims to have shown that when any part of the mesoderm 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

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 interpretation, 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 presumptive 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 transforming action. If the whole regional determination is to be attributed to quantitative variations in so few substances the responsibility 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 specification 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 (19486) 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 reduced edition of the whole. We have seen an example of this in Nieuwkoop’s finding (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 (19436). He cut out the blastopore lip region from a young gastrula of Triton and treated it with alkaline 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 demonstrated 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 selfindividuation.

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 are 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 presumptive neural plate, which at the same time is streaming backwards above it towards the blastopore. Nothing very definite is known about the significance of these movements for the processes of regionalisation, but it seems almost impossible to believe 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 quantitative variation in inducing capacity which all authors seem to agree must exist within the mesoderm. If an evocating substance begins to be released in the mesoderm at the time this invaginates through the blastopore, by the late gastrula stage it would presumably have attained a higher concentration 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 19404, Waddington and Yao 1950) (Fig. 20.28).

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 telecephalon, diencephalon, mesencephalon and myelencephalon. (From Lehmann 1948.) 466 PRINCIPLES OF EMBRYOLOGY

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 presumptive neural plate, which has been moving down towards the blastopore, 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 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 development, 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 with 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 ectoderm 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 individuation. 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 find ourselves forced to appeal to the mysterious 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 ascertainable 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 s 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 1950b, Stern 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 utilise 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.

x * * * *

We shall not cease from exploration And the end of all our exploring Will be to arrive where we started And know the place for the first time.

From Little Gidding by T. S. Eliot.