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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 XIV Regeneration

The word ‘regeneration’ is used to refer to the processes by which an animal restores, or tends to restore, any regions which may be removed. It covers a wide range of phenomena (General Review: A. E. Needham 1952). At one extreme an adult mammal which has suffered the loss of a small part, such as a finger, or a larger part, such as a limb, can do no more in the way of regeneration than merely repair the wound and close the cut surface. At the other extreme a very small part of the normal body of a coclenterate, a flat worm, or a starfish, can restore the whole large region which is missing and become a complete individual. There are all grades in between these two extremes. The power of regeneration is in general greater in lower forms and less in more highly evolved ones, but this rule is only very rough, and when one looks at the matter in more detail it becomes apparent that the capacity for regencration is distributed in a rather arbitrary manner throughout the animal kingdom. Often even closely related forms differ considerably in their regenerative powers. The mass of information on the subject is very large and there would not be space here to review it completely. It is, however, necessary to take a glance at certain aspects of the subject, particularly because of the light it throws on two general points of embryological theory. These are, firstly, the reversibility of determination, and secondly, the field theory.


Workers on regeneration (particularly Morgan in 1901), have distinguished two different modes in which the phenomenon can occut. If a part is removed from some organism, what is left may remain unaltered and regeneration be effected by the outgrowth of a new mass of tissue which becomes modelled into the missing parts. Such a process was called epimorphosis by Morgan. The word ‘regeneration’ is sometimes used in a narrow sense to refer to it alone (e.g. by Child, who employs ‘reconstitution’ as the more general term). In the other mode of regeneration, the part of the organism which is left after the amputation itself becomes remodelled so as to be transformed from a part into a whole organism. To this Morgan gave the name ‘morphallaxis’ which is still in common use.


Epimorphosis can certainly occur with little or no sign of morphallaxis. It does so, for instance, when a young newt regenerates an amputated limb, or an earthworm an amputated head, but it is not quite so certain that morphallaxis ever occurs without some preliminary epimorphosis. For instance, ifa flat worm is cut transversely in half some distance behind the pharynx the posterior half will eventually become transferred into a complete worm and this involves the appearance of a new pharynx some distance posterior to the cut. But it is probable that the first step is the appearance of a small outgrowth from the cut edge and that it is only after this has formed a new anterior part of the worm by epimorphosis that morphallaxis begins to occur and to cause the remodelling of the rest of the posterior portion.


Regeneration is often incomplete, less being formed than is necessary to replace what has been lost. For instance, a regenerated leg in the salamander may lack one or two of the toes, or the regenerated tail in a lizard show defects in its bony structure. There is perhaps nothing very surprising in this. It is more astonishing that animals should be able to regenerate at all than that they should sometimes fail to do so perfectly. It is more unexpected to find that there are also cases of super-regeneration. Itis by no means uncommon for a missing part to be restored in duplicate. In some ways very closely connected with this is a phenomenon which can be regarded as regeneration without any previous loss to account for it. This is the process known as ‘budding’ in which a group of cells at some point in a complete normal animal suddenly start proliferating and develop into a new individual. The process occurs in groups such as the coelenterates and ascidians, when it gives rise to colonies of individuals which often remain closely attached to one another. In some worms, too, a head begins to form in the posterior region of the body and eventually the whole hind end breaks away and becomes a new individual.

1. The origin of regeneration cells and their potentialities

When a part of an animal is removed there are three sources from which the cells which build up the regenerate may be derived: (1) the tissues of the stump may grow out and form the new organ, retaining their original histological character and altering only in so far as they become part of the new region of the body (we shall use the word ‘stump’ in a general sense, to mean the region in the immediate neighbourhood of the place from which the organ has been removed); (2) the body may contain a reserve of undifferentiated or embryonic cells which accumulate at the wound and then differentiate into the tissues of the regenerate; (3) some of the already differentiated tissues of the stump may lose their differentiation and return to a more plastic condition from which they are able to redifferentiate into the specialised tissues of the regenerate, suffering during the process a greater or lesser change in their histological 304 PRINCIPLES OF EMBRYOLOGY

character. The mass of cells which gather at the position of the wound, and which eventually develop into the regenerated part, is known as the ‘regeneration blastema’.

It is probable that in morphallaxis the first process plays an important role. In general, however, it is by no means the most important of the three. Even in animals such as coelenterates, flatworms and oligochaetes in which morphallaxis occurs to a considerable extent, it has been shown that the body contains a supply of undifferentiated cells (known as reserve cells or neoblasts) which play a large part in the regeneration (Fig. 14.1). The stimulus of the wound activates these cells. In the coclenterates those



FIGURE 14.1

Longitudinal section through the gastric region of Hydra, with endoderm

on left and ectoderm on right and the mesoglea (Mes) between. A group of

neoblasts or ‘interstitial cells’ is shown at C.i. At C.ep.1 similar cells are

developing into epidermic (=ectodermal) cells, at C.n.1 into cnidoblasts, and at C.b. into endoderm. (From Stephan-Dubois 1951.)

in the neighbourhood of the wound begin to divide and to produce the tissue out of which the regenerate is formed, while in flatworms the neoblasts even from further away migrate to the wound surface and give rise to the blastema (Stéphan-Dubois 1951). In higher animals such as vertebrates, the direct outgrowth of the already existing tissues is only a minor factor in regeneration. However, the nerve supply of a regenerate is probably always produced in this way, by the sprouting and outgrowth of the nerve fibres in the stump.

There has been a great deal of discussion about the origin and potentialities of regeneration cells in vertebrates, and there is still by no means REGENERATION 305

full agreement on all points. It may be pointed out in the first place that the regeneration process falls into three fairly distinct phases; (i) initiation of the process, and accumulation of a blastema; (ii) growth of the blastema, and (iii) differentiation. These phases can be differentially affected by various means. For instance, Lehmann and Bretscher (1952) found that certain amino-ketones inhibit the first phase (as does x-raying), while colchicine affects mainly the second, and quinoxaline derivatives the third.

The most important aspect of the initiation process is the production of the apparently undifferentiated cells which make up the blastema. This is usually a response to the injury involved in the amputation, but it can sometimes be brought about by means other than wounding. For instance, a number of Russian authors, beginning with Nassonov, have claimed that the formation of supernumerary limbs (by processes which can be considered as equivalent to regeneration) can be stimulated by injecting various tissue extracts and autolysates into otherwise uninjured axolotls, cartilage extracts being particularly effective (see Fedorov 1946). It seems likely that similar substances, released from the injured cells at the surface of the wound, are important in initiating blastema formation in the normal case. In adult Anura, under ordinary circumstances regeneration is either very slight or does not occur at all; and this seems to be due to an incapacity of the animal to produce a blastema. If a wound surface in such an animal is treated with strong sodium chloride solution (Rose 1944) or is lacerated mechanically (Polezhayev 1946), blastema cells appear and a considerable amount of regeneration may occur. Denervation of a limb in the urodele amphibian, on the other hand, usually leads to a failure of blastema formation and thus of regeneration (cf. Rose 19484).

The next question to consider is whether the blastema cells in vertebrates are derived from reserve undifferentiated cells or from the dedifferentiation of the tissues in the near neighbourhood of the wound. Experiment has now demonstrated that the second of these is by far the most important source of the regenerating cells, although a minor contribution may come from the relatively undifferentiated connective tissues near the cut. The local origin of the cells can be demonstrated as follows: A limb is removed from a normal diploid urodele and a similar limb from a haploid specimen of the same species is grafted into its place. After union is complete the limb is amputated again, leaving only a smali segment of the haploid limb attached to the stump. The regenerated limb is then found to be haploid and must have been derived from the cells of the small haploid segment (Hertwig 1927). Again it is a very general observation that irradiation with x-rays decreases an animal’s capacity to regenerate 306 PRINCIPLES OF EMBRYOLOGY

(cf. Brunst 1950), chiefly because it produces chromosome rearrangements which, when mitosis occurs, lead to unbalanced and inviable nuclear constitutions. Thus x-radiation can knock out the reserve cells of a planarian while leaving the animal as a whole capable of living but no longer of regenerating. If only the anterior region of a worm is irradiated and then a part of this region removed, regeneration is delayed for the time that it is necessary for the reserve cells to move from the nonirradiated region through to the cut surface (Wolff and Dubois 1948). Experiments of this kind in newts have given no evidence of any extensive movement of cells to form the regeneration bud in vertebrates.

It appears therefore that a regenerated organ in a vertebrate is in the main constructed out of dedifferentiated cells from the tissues close to the cut surface. New cells accumulate on the surface in a densely packed group to form a regeneration bud or blastema. Histological examination of the process gives evidence that muscles cells, bone-forming cells and other mesodermal elements contribute to the undifferentiated mass (cf. Manner 1953). Recent studies by Rose (19486) also suggest that dedifferentiated epidermal cells make a considerable contribution. In the blastema all the cells lose their characteristic histological appearance. The important question arises whether this is a true dedifferentiation, and the cells able to develop again into something other than they were originally, or whether it is a deceptive appearance similar to that which can be seen in tissue culture, where cells from differentiated tissues lose their characteristic appearance and make an impression of being dedifferentiated, but in reality retain their specific nature and can develop again only into what they were before. The evidence suggests that to some extent at least the formation of a regeneration blastema involves a true dedifferentiation.

Perhaps the most conclusive evidence for the occurrence of true metaplasia (a change of character from one differentiated type to another) comes from the rather special case of the so-called Wolffian regeneration of the lens (Reyer 1954, J. Needham 1942). If the lens is removed from the eye of an amphibian the edge of the retina begins to grow and forms a group of cells which differentiate into a new lens replacing the old one. It seems quite clear here that differentiated retinal cells change their character completely to give rise to the final lens. This very odd type of regeneration has been known since the ‘nineties of the last century when it was discovered by G. Wolff, after whom it is named. The retina does not need to suffer any wounding to become stimulated to start regenerating; this occurs as soon as the lens is removed even if the retina is quite uninjured in the process. It has been clearly demonstrated that the stimulus is chemical. The lens apparently gives off some substance which holds the retina in check, and regeneration begins as soon as this is removed. Within the retina there is a gradient in readiness to undergo regeneration, the dorsal region showing greatest capacity and the ventral the least.


The situation is not so clear in the more usual types of regeneration, such as that of the limb or tail. It has been claimed by many authors (e.g. by Weiss 1930), that the blastema is at first quite undetermined and is competent to develop into almost any part. The first part of this claim seems to bejustified. For instance, when early blastemas from regenerating limbs are transplanted into an indifferent situation, such as the body cavity of an adult salamander, they form masses of undifferentiated cells, which appear similar to malignant tumours (Waddington 1940); it is probable, however, that they do not become fully malignant but are eventually encapsulated and keratinised. If a similar blastema is transplanted from a limb to the cut surface of an amputated tail, Weiss originally claimed that it would become determined by its new surroundings and thus differentiate into a tail; but this interpretation has been challenged on the grounds that it is difficult to be certain that the transplanted blastema was not simply resorbed, the tails which eventually appeared being formed not from grafted tissues but by the stump of the tail in the normal way. It would seem that the question could be settled by using polyploid or other specifically recognisable tissues to provide the transplant, but this has not yet been successful; May (1952), who tried it, found it impossible to recognise the cells of a triploid transplant in the redifferentiating tissues of the regenerate. Most authors who have reviewed the subject recently (c.g. J. Needham 1942, A. E. Needham 1952) reach the conclusion that the weight of the evidence is against the possibility of changing the fate of the blastema from one organ to another.


In contrast with this, there is a good deal of evidence that cells of the blastema can differentiate into any of a number of different types of tissue. Thus the power of regeneration can be removed from an amphibian tail or limb by x-raying; then one particular type of tissue from a normal limb can be grafted into it, and a few days later the limb amputated in the region of the graft. Luther (1948) claims that under these conditions, leg "skin transplanted to an x-rayed tail gives rise to blastema cells which form all the tissues of the appendage (muscle, bone, blood vessels, etc.) but that they showed a tendency to form legs instead of tails. Trampusch (1951) has described similar evidence of changes of tissue specificity following transplantation of healthy skin, muscle or skeletal tissues to irradiated limbs. A converse type of evidence was obtained at a much earlier date by Weiss (cf. 1930), who showed that if one of the long bones is removed from a limb, which is then amputated through the defective region, the regenerate will be provided with bone, although the bone will not be replaced in the region of the old limb from which it was excised (but this situation does not seem to hold for the tail, since if the axial organs are removed from that organ, amputation is followed by the regeneration of a structure with the same defect as the stump [Vogt 193 t]). Finally, Schotté (1940) has claimed that if a very young regeneration blastema is transplanted into a situation in which it is exposed to the appropriate inducers, it may be caused to differentiate into lens or ear tissue.


We scem therefore to be driven to the rather unexpected conclusion that, although the cells of the early blastema are very labile in their histological properties, and can become almost any tissue (except probably nerve), there is little evidence that they can change their organspecificity. If this is so, then, for instance, tail epidermis can undergo a ‘metaplasia’ by which it becomes converted into muscle—but it will be tail muscle even if the process occurs after transplantation to a site on the limb. Since organ-specificity is a rather unfamiliar concept, and we have no clue as to its chemical basis, it is surprising to find it obtruding itself in such a definite manner in experiment. There are, however, other contexts in which it appears that organ-specificity is a rather distinct character in the later stages of development. For instance, in birds the mesodermal core of a feather papilla can induce epidermis to develop into a feather germ; and the type of feather eventually produced depends strictly on the region of the body from which the competent epidermis comes, breast epidermis always forming breast feathers, saddlee pidermis saddle feathers and so on (Wang 1943).


The evidences of histological metaplasia are of fundamental importance for our understanding of the process of determination. They show that determination which occurs in embryonic stages, and the high degree of histological differentiation which follows it, need not involve absolutely irreversible changes, although shortly after the period of embryonic determination no means are known to bring the cells back to a plastic condition. Later in life the stimulus of wounding may have this effect.’ It follows that those nuclear genes which are concerned in the differentiation of the new histological type to which the cells switch over, must have persisted throughout the earlier period. If the undifferentiated blastema cells are capable of redifferentiating into any and all adult tissues, one would have to conclude that the whole of the genotype was still available and that there had been no irreversible inactivation of genes during development. The evidence does not yet go quite so far as this, since we still do not know the full range of capacities of blastema cells, but it seems to be tending in this direction.

2. Field action in regeneration

The process of regeneration usually restores exactly what is missing to complete a normal individual. That means that the growth and differentiation of the material is reP*ed in the first place to the stump or remaining part of the animal, arin the second to the final complete form. It was this situation, more than any other, which has tempted biologists to employ the concept of ‘fields’, and regeneration provides the classical context for a discussion of the validity and meaning of this notion. We shall find that so long as it is not taken to provide a solution to the problems, but rather as an enlightening way of formulating them, the field concept can be very useful. Even those cases, known as ‘heteromorphoses’, in which regeneration does not restore normality, can be usefully discussed in such terms.

There is not space here to discuss regeneration fields in all the groups in which they occur, and we shall limit our attention to certain aspects of the process in coelenterates, platyhelminths and vertebrates, An introduction to the literature, including that concerning other groups, may be found in A. E. Needham (1952).

(a) Coelenterates

During the eighteenth century, the experiments of Trembley of Geneva on the regeneration of H: 'ydra made this topic, for a time, a fashionable diversion in the drawing-rooms of the elegant. Work has continued on it ever since. In recent times, the elongated marine hydroids, such as Obelia, Tubularia, Corymorpha, etc., have been more commonly employed as experimental material (General Reviews: Barth 1940, Child 1941).

The power of regeneration of all these organisins is exceedingly great. The animals consist of an apical region, the hydranth, which is provided with tentacles surrounding the mouth; the main body, containing a gastric cavity; and a foot or stolon region, by which the animal or colony is attached to the substratum and which does not contain any gastric space. The marine forms which have been mainly studied occur as colonies, consisting of many individual polyps united by their gastric portions. These regions are known as the coenosarc, and in many forms are enclosed in a hard chitinous sheath, the perisarc.

Any fragment of the gastric or coenosarc region, which is large enough not to fall to pieces as a result of the wounds inflicted in isolating it, can produce a new hydranth by regeneration; usually it also produces a new stolon. Most attention has been paid recenitly te the rate and frequency of the regeneration of the distal end, which gives rise to a new hydranth. This is influenced both by the level from which the fragment is taken, that is the distance behind the original hydranth, and by whether a hydranth remains attached to the fragment or not.

Suppose a piece of Tubularia or some similar form is taken, consisting of a hydranth attached to an unbranched ht gth of coenosarc. This is cut in two at some level, i.e. at some definiten.astance not too far behind the hydranth. The usual result is that a new hydranth is formed at the distal end of the proximal piece, but none appears at the proximal end of the distal fragment to which the hydranth is still attached. If, however, in another similar piece, the cut is made very slightly nearer the hydranth, a similar result occurs; and now the hydranth formed on the proximal piece is arising in the very region which, in the previous experiment, failed to form hydranth. Thus this failure must have been due to its having been still in continuity with the original hydranth, and not to any inherent lack of power of regeneration. This phenomenon, in which the presence of a hydranth suppresses the regeneration of a second hydranth at the other end of a fragment, is spoken of as dominance of the hydranth. (There should be no temptation, of course, to confuse this use of the word with that current in genetics.) (Fig. 14.2.)

The dominance of an apical hydranth gradually diminishes along the length of the fragment, and at the proximal end of very long fragments is hardly appreciable. This suggests that the hydranth sets up a high level of some activity, or concentration of some substance, which falls off away from it in a gradient along the coenosarc. Rather surprisingly at first sight, there is no evidence of dominance in very short fragments, which tend to regenerate hydranths on the proximal surface as well as the distal. This can, however, find an explanation if we suppose that dominance only occurs if there is a considerable difference in activity between the two ends of the fragment and that when the isolate is very short, the difference is so small as to be ineffective.

One cannot accurately measure the degree of dominance in longer pieces without taking into account another factor which varies along the length of the coenosarc. Experiment shows that, quite apart fron dominance, the inherent rate of regeneration falls off as the distance from the original hydranth increases. Barth (see 1940) investigated this by making a series of equal-sized small isolates from the different levels of the stem. Any phenomenon of dominance within the fragments was prevented by constricting them tightly in the middle by a ligature which effectively isolated the two ends from each other. Both ends then produce hydranths. REGENERATION 311

The rate of formation of the hydranths falls off from the distal to the proximal fragments, and this gives a measure of the gradient in intrinsic regeneration rate, independent of dominance (Fig. 14.2).

In order to estimate dominance, a fragment of medium length is taken and allowed to regenerate at the proximal end; and its rate of regeneration is compared with that in a similar fragment in which the influence of the original hydranth has been suppressed by a ligature just behind it. Again, if a piece of coenosarc is isolated, the original hydranth being discarded, the distal end will regenerate more rapidly than the proximal, and the distal regenerating hydranth will exert some dominance over the proximal one. The degree of dominance can be measured in the same sort of way as before, by comparing the rate of proximal regeneration when the distal end is left free or is ligatured.




FIGURE 14.2

Figure a, rates of regeneration (in » per hour) at various levels along a stem of Tubularia; this is the inherent rate, any interference by dominance being avoided by ligaturing; b, c, d, e, showing bipolar regeneration in the shortest lengths, then complete dominance of the distal end, then partial dominance, which weakens in the longest lengths; f; regeneration at the proximal end is rapid when a ligature prevents the distal end exerting dominance, but slower (g) when dominance is possible. (From Barth 1940.)


These experiments reveal the existence of two gradients, one of intrinsic regeneration capacity and one of dominance. Since the hydroid stem is a linear structure, we are only confronted with gradients in one dimension, along it. We can take them as being the simplest possible instances of fields, which in other cases will usually extend in two or three dimensions. The two gradients obviously correspond to the two types of fields mentioned on p. 25; the gradient of intrinsic regeneration rate is a ‘field’ of competence, that of dominance is an expression of a hydranth individuation field which extends outside the limits of the actual hydranth structure.


It must be clearly recognised that when we speak in this case of gradients or of fields, we are doing no more than describe the phenomena revealed in experiment, and are still far from a satisfying explanation of the mechanisms involved. We can, however, in the case of the hydroids, make some further progress towards this, both in the refinement of the theoretical formulation of the case and in the experimental discovery of new facts about it.


Let us consider the theoretical aspects first. Both Barth (1940) and Spiegelman (1945) have suggested that the mechanism of the individuation field is to be found in a competition between the various regions for physiologically necessary substances. Suppose, for instance, that the production of a new hydranth involves the transformation of some raw material K into hydranth material R. Then clearly if two hydranths are being formed, and are physiologically connected so that they both utilise a common supply of K, the development of one hydranth will tend to inhibit that of the other. If one hydranth gets some sort of start over the other, or is more effective in drawing supplies of K from the pool, then it will be dominant, and, while being little inhibited itself, will have a strong depressant effect on the other.


If one makes assumptions about the character of the reactions, one can put the situation into mathematical language. One of the simplest hypotheses, which is adopted by Spiegelman in his formulation, is that the production of R is (i) proportional to the amount of raw material still available, i.e. to K — R; and (ii) the efficiency of utilising the raw material is reduced as time goes on and the concentration of R builds up. The second assumption could be expressed, to a first approximation, by supposing that the rate of formation of R is proportional to b — cR, where ¢ is some constant.


For a single process of hydranth formation we should then have an equation of the form aR (K — R)(b — cR). dt If there are two competing sites of hydranth formation, we shall have to consider an R, a b and ac for each of them, which we may indicate as Ry, Re, by, be, €1, €g. Moreover in accordance with the second assumption above, we must expect that the formation of hydranth at one site has an effect on the efficiency of hydranth production not only at that site but at the other one also. We can cater for this by including a new term in the last bracket of the equation as given above. Thus for two competing hydranths, we shall have to consider equations of the form

dR. = (K — R,)(b: —GR, — 42Rs) and dR rd = (K — R,)(b: — Re — dy Ri}

One can see, in a general way, the results which processes of this kind would produce, by considering the situation which would arise when the processes had gone to completion, by which time no further change would be occurring, and the (dR/dt)s would be zero. Then we shall have

by — oR, — dR, = 0, and bs ane Re coe do,R, = 0. From the first of these equations, we sec that, in this final state

pn take

q Q


Since if there had been no competition (i.e. if d,, were zero), Ry would

have been it is obvious that the competition has led to some degree of 1 inhibition of R,; and the same is of course true of R,. Also dominance will occur when either R, or R, is larger than the other; and this may happen either because of the relations between the intrinsic efficiency constants b or on account of the ‘interaction’ coefficients c and d, or from certain combinations of these. For instance, if the interaction coefficients are the same for all sites, but there is a gradient in intrinsic efficiencies we shall have cR, = b, — dR, and cR, = b, — dR,, whence it is easy to show that R,-R, = by — bs aa)

so that if b is greater than b,, R, will be larger than Rg, and there will be dominance of the site with greater efficiency over that with less. It is clear, without our going into the details of the other possible cases (see Spiegelman 1945), that the assumption of physiological competition does provide a mechanism by which gradients in efficiencies of synthesis or interaction would give rise to phenomena such as dominance. It thus makes it possible to envisage field phenomena in a form in which they become amenable to physiological analysis, aimed for instance at measuring the relevant efficiencies or discovering what substances are being competed for, etc.


As to the ‘substances’ involved (we have to use the word in a broad sense), we have two main pieces of information, which have not yet been fully brought into relation with each other. The first to be discovered was that oxygen is highly important. It probably operates in two ways, firstly as a component of the stimulus which sets the regeneration going, and secondly as one of the reactants while the process is proceeding (Barth 1940). Its importance as a stimulus can be demonstrated if a hydranth is cut off and the perisarc pulled forward and ligatured in front of the cut surface, so as to shut it off from the surrounding sea water; no regeneration takes place. The same result can be obtained by covering the cut surface with a piece of glass tube. Moreover, if the perisarc is cut open so as to expose a region of the coenosarc, regeneration may occur, particularly in oxygen-rich water, even if no wound has been inflicted; and the injection of a bubble of oxygen between the perisarc and coenosarc may have the same effect. The continuing importance of oxygen during the whole process of regeneration is shown by the fact that the rate of formation of new hydranth is highly dependent on the amount of oxygen in solution in the water.


Child, and many workers following his lead, have been emphasising for some time the importance of gradients of respiratory rate which they claimed to demonstrate in hydroids and many other regenerating animals, and in eggs in which field phenomena play a leading role (see Child 1941). It was claimed that field processes always depend on gradients in metabolic activity, and that the metabolic activity which is most crucial is that of respiration. The expression ‘metabolic activity’ is, of course, so general that, in those terms, the hypothesis is little more than a truism; a field must obviously have graded differences between its parts, and the portmanteau phrase “metabolic activity’ could cover these whatever their nature. The part of the theory which it is important to discuss is, therefore, the notion that it is respiration which is basic. Extended discussions will be found in Child’s own book (pro) and in Needham 1931 (pp. 582 ff.) and Brachet 1945 (contra). The general sense of the situation would seem to be that, whereas various indirect methods (e.g. susceptibility to poisons, reaction with vital dyes, etc.) often give evidence for the existence of some sort of gradient, it is by no means clear in most cases that the gradients primarily affect respiration; and moreover it remains obscure whether the gradient of respiration, if there is one, is the causative basis of the observed field or rather merely another expression of it. The two most crucial embryological cases in which critical evidence might be sought are those of the echinoderm animal-vegetative gradients and the amphibian organiser; in neither of them is a gradient in respiratory rate of demonstrable importance (see pp. 88, 200).


In the hydroids, the situation is somewhat more favourable to Child’s ideas, since there is fairly good evidence that a gradient in rate of respiration actually exists. In the early measurements of the oxygen uptake of fragments taken at successive levels behind the hydranth, the experiments lasted so long that an important amount of regeneration would have occurred, and the gradient of rate found, with the hydranth end respiring fastest, may have been mainly an expression of the faster rate of regeneration of this end. But Barth (see 1940) has made accurate measurements over short periods, and finds that there is almost certainly a gradient in this sense in the hydroid immediately after cutting. This, however, does not by any means necessarily imply that the respiratory gradient is a cause, and not a mere concomitant of the gradient in regeneration rate.


Turning now to a type of ‘substance’ quite othér than oxygen, it seems that considerable importance should be attached to the finding of Tardent (1954) that there is a gradient in the concentration of neoblasts or regeneration cells (known also as interstitial cells). This was found both in Hydra and Tubularia (Fig. 14.3). These cells increase in number near the cut surface, but it is not yet clear to what extent this is due simply to multiplication or how far migration from other parts of the animal is involved. If the latter process is extensive, it may be that they are the most important ‘substance’ (if one can call them that) for which the two ends of an isolated length of hydroid are competing. It would be very interesting to know whether their rate of respiration is higher than that of the other tissue cells, in which case the respiratory gradient described by Child and Barth might be a reflection of the gradient in ncoblasts; but there is as yet no definite evidence on this point.


It seems probable that when a hydranth is removed from a hydroid, the gradient from the new distal end is established in two stages. First there must be an accumulation of neoblasts at the wound surface, presumably as a response to the increased availability of oxygen. But after the initiation of the hypostome or mouth region of the new hydranth, this may be itself responsible for building up the gradient. Several authors have shown that this region, when transplanted into the side of another polyp, is particularly efficient at causing the production of a new hydranth in its neighbourhood, much of which is formed out of tissues of the host (Beadle and Booth 1938, Yao 1945). This is a typical example of an ‘assimilative induction’, in which the transplant acts as the carrier of a powerful individuation field. In Hydra, whose powers of regeneration are more moderate, only the hypostome can induce a new hydranth in this way. In Tubularia, on the other hand, as we have seen, mere laceration, or the injection of a bubble of oxygen, will suffice to produce the same result. In such extremely regenerative forms, the tissues can be cut up finely and reduced to a homogeneous mass, and are still capable of giving rise to a well-organised hydranth from the upper surface where the availability of oxygen is greatest (cf. Barth 1940).




FIGURE 14.3

Frequency of interstitial cells along the stem of Tubularia. (After Tardent 1952.)

It is worth noting, as a curious and so far unexplained fact, that a hydranth which is actively in the process of regeneration is not able to restore itself if injured before the process is complete (Davidson and Berrill 1948). The regenerative capacity appears rather suddenly just when the new hydranth reaches its final functional histological state; and it will then not only react by regeneration to any subsequent injury, but will replace parts which have been removed during its development. This may perhaps mean that during the first regeneration all the neoblasts are involved in the process and there are no more available to deal with a second injury, but the matter has not been fully investigated. It is commonly found that embryos in the most active phases of development have less power of regeneration than at either earlier or later more quiescent periods. Thus in the Amphibia the early gastrula can easily repair defects, and so can the young tadpole, but the neurula or early tail-bud stage has little capacity for regulation.

(b) Flatworms

It will be useful to supplement the account of regeneration fields in hydroids by a somewhat shorter discussion of similar phenomena in flat worms, which are also a lowly group of invertebrates but somewhat more highly evolved than the coelenterates (General Reviews: Bronsted 19544, b, Wolff 1953). We shall find that in the flatworms the active individuation field in the adult is less powerful than that which is maintained by the hydranths in hydroids. As a consequence of this the main role in directing the early course of regeneration is played not by the adult organs which remain in the regenerating piece, but rather by a static field of regeneration potential, which can be compared with the gradient in regeneration rate which was characteristic of the hydroid stem. This determines the character of the blastema which is formed, and that in its turn then brings about the development of the appropriate organs.


Regeneration has been mainly studied in triclads, of which Planaria is a characteristic genus. The whole group are frequently referred to as planarians. Ifa planarian is cut in two by a transverse cut it is frequently found that the anterior segment regenerates a tail and the posterior segment a head. In both cases the process starts by the formation of a blastema, which is produced by neoblasts which migrate to the wound surface (see p. 306). Many species of planarians are provided with eyes in the anterior region of the head. One of the first signs of head regeneration is the appearance of such eyes in the blastema. This occurs at an early stage, even if a large part of the anterior of the worm has been removed. Thus it seems that in regeneration of the head the most anterior part is formed first and whatever else is required is, as it were, intercalated between this anterior part of the head and the remaining posterior end of the body. During the later stages of regeneration, however, a good deal of morphallaxis occurs, that is to say a remodelling of the original posterior part of the body.


The occurrence of these easily recognised organs, such as eyes, makes it simpler to study the regeneration of the head than that of the tail, and most work has concerned itself with this type of regeneration. The simplest experiment consists in placing the transverse cut, which divides the worm in two, at various levels from the anterior to the posterior. We find that, as was the case in the hydroids, any particular level of the body may form either a head or a tail according to whether it is attached to an posterior or to a anterior piece. However, the ability to regenerate a head, as measured either by the frequency with which this is successfully accomplished or by the time taken to do so, is not usually constant along the whole length of the worm. Different species fall into a number of classes in this respect. There are some in which no regeneration of a head occurs from any part; in others, of which the well-known Dendrocoelum lacteum is one, the anterior end regenerates easily but the ability to form a head falls off rapidly and has disappeared at the level of the pharynx. In others, the curve expressing the ability to regenerate a head (the so-called ‘headfrequency curve’) falls off more gradually and even the most posterior end of the body sometimes carries out the regeneration successfully. Some Planaria normally reproduce by transverse fission, a new animal forming in the posterior end of the body of an old one. In these the head-frequency curve, after falling in the region of the pharynx, rises again towards the posterior end. Finally there are some species, such as Planaria velata, in which the ability to regenerate a head appears to be equal along the whole length of the body. There are also tail-frequency curves in all these species, but less is known about them.


The ability to regenerate a head is, however, not fully expressed merely by a curve which assigns some definite ability to each body level. There is, in point of fact, a gradient from the midline of the body towards the margins as well as from anterior to posterior (Fig. 14.4). We have to deal with a two-dimensional field of head-forming ability rather than with a one-dimensional gradient of it. The type of regeneration which occurs when the planarian body is cut in more complicated ways can usually be deduced fairly simply from the principle that in posterior pieces a head forms at that point of the cut surface which has the highest value in the head-producing field. To account for the fact that the same point would form a tail if attached to the anterior piece, the simplest assumption seems to be that the regeneration tends to occur in such a way as to carry on the gradient which is already intrinsically present within the fragment. In this way one can understand such peculiar phenomena as the appearance of the two heads at the shoulders of the T-shaped cut shown in Fig. 14.5A-D. The appearance of heads in the situation shown in Figs. 14.5E and F is not so fully accounted for, since here the regenerating edge is connected directly both with the anterior and the posterior parts of the body, and further subsidiary hypotheses would be necessary before it was clear what we might expect to obtain. There seems at present to be no adequately tested hypothesis which can deal satisfactorily with all the numerous and complicated experiments which have been made in this field.


It is important to note that it is only regions of the old body in the immediate neighbourhood of the cut which exert an influence on the course of regeneration. Bronsted (1939) has isolated a section of the body anterior to the pharynx by two transverse cuts (in Euplanaria Iugubris) and then grafted the anterior tip containing the original head with reversed polarity to the posterior edge of the isolated segment. The grafted head in this situation exerted no apparent influence on the regeneration at the anteriorcut surface, which proceeded to form a head exactly as it would have done normally. Similarly, if a window is cut in a planarian, as in Fig. 14.4, the existing old head does not inhibit the regeneration of the new head in the window. Again, Raven and Mighorst (1948) have shown that the rate of head formation at a given level in Euplanaria Ingubris is not in the slightest affected by the presence or absence of a further posterior cut at which, if it is present, a tail will simultaneously be regenerating. Thus in planarians there is little or no evidence of competitive interaction between the two ends of an isolated segment, or between a regenerating surface and already existing organs. We have thus little sign of anything corresponding to the phenomenon of dominance in hydroid regeneration.


FIGURE 14.4

On the left, the head-regeneration field in the planarian Bdellocephala punctata. On the right, a head regenerated in a ‘window’ cut in the anterior region. (From Bronsted 1946.)


There are, however, some inductive interactions in operation in planarian regeneration. If the head region of Dugesia Ingubris is grafted into the posterior of the body, behind the pharynx, it can induce the formation of a supernumerary pharyngeal region from its neighbourhood; while in Polycelis nigra, the cerebral ganglion induces the formation of eyes though only from a limited competent area which extends from the anterior to the level of the pharynx (work of Sengel and Lender, see review of Wolff 1953). Wolff has summarised the conclusions from these experiments in a diagram which is reproduced as Fig. 14.6. The first event in a blastema engaged in head regeneration is the formation of the ganglion; this induces a cephalic region (which in many forms includes eyes); this region induces a pre-pharyngeal region, and that again a pharyngeal region; and in the Jatter the pharynx itself eventually appears. These inductive relations can be regarded as the expressions of an individuation field which is most powerful when it arises within a blastema, since it can then affect even the anterior part of the body, but which persists at a lower intensity even in the fully developed organism.


FIGURE 14.5

A, B, C, D, stages in the regeneration of two heads, following the T-shaped cut shown in A; E, F, regeneration of a head following a longitudinal cut. (After Beissenhirtz, from Bronsted 1946.)





FIGURE 14.6

Successive inductions during the regeneration of planarians. After a poster ior region is isolated (a) the first step is the formation of a ganglion (C) in the blastema (b). This induces a head (T) provided with eyes (y). The head region then (d) induces a pre-pharyngeal zone (Pr), and that in turn (e, f) a pharyngeal zone (Ph), in which (g) a pharynx finally appears. (From Wolft 1953.)

(c) Amphibia

Larval and adult urodele Amphibia (newts, salamanders, axolotls, etc.) can regenerate legs or tails and other organs fairly readily (General Reviews: J. Needham 1942, A. E. Needham 1952). The capacity of Anura (frogs, toads, etc.) is much less in this respect, the power of regeneration usually being lost at about the time of metamorphosis. The regenerative phenomena in urodeles provide a very good example of one kind of field action. It was in fact in this connection that the concept was first extensively discussed by Weiss, one of those who introduced the notion of fields into embryology, and who contributed greatly to the early experimental work on vertebrate regeneration (see Weiss 1930, 1939). As we shall sce, however, the field which is operative in amphibian regeneration is strictly an individuation field concerned with the building up of a complete unit, and shows no sign of activity outside the limits of this unit. There is therefore nothing which corresponds to the dominance of the old hydranth in the regeneration of hydroids.


As far as regeneration is concerned the urodele body can be considered to be made up of a series of organ-districts. If a complete district is removed, it cannot be regenerated. If, however, any part of the district is eliminated regeneration will restore what is missing, unless indeed for some reason the blastema is smaller than normal, when a deficient organ may be produced. The limit of the organ district of the tail, for instance, is the last sacral vertebra. If the tail is amputated anterior to this, no regeneration occurs, whereas if the cut is made anywhere further posteriorly, a complete tail is formed. Regeneration occurs strictly within each organ district and is uninfluenced by the position of that district within the body as a whole. Thus if a limb is transplanted to the middle of the back and then amputated, the stump nevertheless regenerates a limb. The character of the regenerate, in fact, depends on the tissue in the immediate neighbouthood of the wound. If a hindlimb is amputated, a forelimb transplanted to the stump, and then the forelimb again amputated so as to leave only a small section of it, the regenerate will still be a forelimb and show no influence of the hindlimb stump, which is further away from the wound surface.


Amphibian regeneration exhibits several peculiar polarity phenomena. In the first place it should be noted that the regenerate always produces the parts distal to the wound surface and apparently never those proximal to it. Thus if a deep V-shaped cut is made into a limb, both faces of the wound may produce a regenerate and both of these will develop into the regions of the limb which should lie distal to the cut. Again, if a segment of a limb, say the region near the knee joint, is isolated by two cuts and then inserted into the body-wall in such a way that the proximal surface as well as the distal can regenerate, it will be found that the proximal surface does not form a new femur and upper part of the limb, but that both surfaces form the distal extremity. J. Needham (1942) accepted some earlier data which suggested that in such cases the proximal surface regenerated only so much of the lower limb as had originally been isolated. This would have been a very peculiar situation, since it would have meant that the individuation field had been permanently altered by the isolation. It appears, however, from more recent work of Monroy (1942), that the regenerate from the proximal surface forms the whole missing region and not only a part of it (Fig. 14.7). We can conclude therefore that regeneration will always produce the whole missing region from the cut surface to the distal tip of the organ, even if this means that a reversal of polarity has to occur at the place where the cut was made. It may be pointed out that if a section of a limb is removed and the distal region grafted back on to the stump, no regeneration of the missing segment occurs, probably because there-is no Opportunity for a blastema to form,


FIGURE 14.7

(Left) Part of an anterior limb of a newt, consisting of sections of theThumer us, and of the radius and ulna, was grafted into the flank. Both ends have regenerated and each blastema has produced all the structures distal to the position of the wound from which it arose. (From Monroy 1942.)

(Right) Two limb-like regenerates (a and 6), and one (c) showing features of both tail and limb, all from axolotl tails onto which limb-skin has been transplanted. (From Luther 1948.)

Regeneration, of the limb at least, is not only limited by the fact that it proceeds only distally and never proximally: it can also not proceed laterally. That is to say, if a limb is split longitudinally and one half removed, this half is not replaced. However, if the half-limb is now amputated regeneration occurs and the regenerated portion of the limb is complete in cross-section. Similarly, if one of the long bones of a limb is removed, it will not be regenerated, but if the limb is now amputated through the boneless region, the regenerate will be provided with the full necessary complement of bones (the rule does not hold for the tail, see page 308). This formation by the regenerate of structures which are more complete than the stump shows clearly that regeneration is not merely a result of the outgrowth of the tissues of the stump, but is a phenomenon in which an individuation field arises in the blastema and leads to the development of the complete section of the organ between the level of the cut and the distal tip. However, although the structure of the regenerate is not directly produced by the outgrowth of the tissues exposed at the cut, those tissues are operative in determining the character of the individuation field which arises. In most cases, as we have seen, a mere absence of a tissue does not lead to any deficiency in the individuation field. If, however, tissues from two different organs are mixed, compound or hybrid individuation fields may appear. Thus Liosner and Woronzowa (1937) and Monroy and Oddo (1943) have found that if muscles from the tail are grafted into the urodele limb at the site of amputation the regenerate may be intermediate in structure between a tail and a limb. Similarly intermediate structures were found by Luther (1948) after the transplantation of skin from the foot to the tail (Fig. 14.7).

Suggested Reading

For a full treatment, A. E. Needham 1952. Other recommended reading: Weiss 1939, pp- 458-478; Barth 1940, Rose 1948); Avel 1940 (oligochaetes, not treated in the text); Spiegelman 1945.


PART TWO

THE FUNDAMENTAL MECHANISMS OF DEVELOPMENT

Ae IN the introductory chapter a list was given of the basic concepts which experimental embryology has developed—those of determination and differentiation, brought about by the mechanisms of ooplasmic segregation, evocation and field action. The accounts of the development of the various classes of animals given in Part One have exhibited the type of facts from which these concepts have been derived. The facts have, for the most part, been obtained by typically biological modes of experiment; for instance, by the transplantation from one place to another of whole cells, or even large masses of cells. The theories to which they have given rise have in consequence also been framed in terms of concepts which belong to the biological realm and cannot be immediately brought into line with the ideas of the more fully developed sciences of physics and chemistry. As Weiss (1947, 19506) in particular has emphasised, we cannot be permanently satisfied with this situation, but must attempt to push our analyses down towards the level at which we are discussing the interactions, combinations and synthetic activities of particular substances. In this connection he has coined the expressive phrase ‘molecular ecology’ and there is no doubt that a fully developed embryology ought to be able to expound the processes of development in terms of the changes in the populations of molecules making up the cells of the different tissues and should not have to rely on concepts such as evocation and competence, which are special to it alone.

The programme which Weiss proposes is, however, a very difficult one and we are still far from the goal which he envisages. Indeed, the distance is so great that there is probably some danger in attempting to cover it in a single step. There is an intermediate level, between that of the organisers, fields, etc. usually considered by embryologists and the ultimate molecular constituents of living matter, which requires to be thoroughly explored before we can feel the ground firm enough under out feet to have any confidence in attempting to frame theories in chemical or physical terms. This is the level which deals with the activities of the different categories of cell constituents. As we have pointed out, experimental embryology has already approached it in several different contexts; for instance, in relation to the cell granules of mosaic eggs (p. 101), the mitochondria of sea-urchins (p. 90) and the microsomes of Amphibia (p. 212). These are all constituents of the cell cytoplasm, not of the nucleus. The crucial role of inductive processes (both between tissues and within embryonic fields) demonstrates that the initial differentials which guide the development of various parts of the egg into different channels depend in many cases on substances which can pass from cell to cell and which must therefore be extra-nuclear. There is another method of approach which has penetrated very deeply into this field of the activities of cell parts, that is the science of genetics which has operated mainly by the study of heredity. The knowledge it has yielded has in the main concerned the effectiveness of the nucleus and the chromosomes, an aspect of cell behaviour about which the methods of experimental embryology have not told us very much. More recently, moreover, genetics has begun to produce most valuable information about cytoplasmic particles, which it knows under the name of ‘plasmagenes’.

It would, of course, be inappropriate to attempt to give even a sketch of all aspects of genetics in a book devoted to embryology, but there is great deal of genetical information which is not merely relevant but which is essential to our purpose. In fact, when we attempt to discuss the fundamental mechanisms of development we are in a region in which the distinction between the sciences of genetics and embryology breaks down. Genetics using its normal method of studying heredity has revealed the existence of genes which control to a large extent the character of the animal which will develop from the fertilised egg; the way in which the genes operate in doing this belongs by definition to embryology. The union of the two sciences could hardly be more intimate.

In the first chapter of this part we shall discuss in broad outline what genetics has revealed about the general nature of the epigenetic system and the way genes are involved in it. This supplements the experimental embryological material, to complete the picture of development at the biological level. We shall then pass on to consider the basic mechanisms of development in terms of the activities of cell constituents, using facts some of which are conventionally considered to belong to genetics, others to embryology. The greater part of this discussion will be concerned with the differentiation of substance which occurs during development; that is to say with the formation of new materials or new types of tissue. The last chapter deals with the other aspect of development, the moulding of tissues into organs of definite shape and form.


   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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