Waddington1956 16

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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 XVI The Activation of Genes by the Cytoplasm

It has frequently been argued that genes control only the later-developed and more superficial characters of animals and that the development of the basic plan of the body is controlled, not by them, but by the cytoplasm of the egg; and this contention has been hotly disputed by geneticists who seem to feel that it disparages the importance of their subject. We realise now that, as in so many such controversies, both sides are in the right. Undoubtedly within any one lifetime a great deal of the basic pattern of the body is dependent on the configuration of the cytoplasm of the egg; one need only remember such phenomena as the arrangement of ooplasms in mosaic eggs (p. 106), of gradients in the echinoderms (p. 85), or of the formation centre in insects (p. 126). There are indeed many cases in which genetic differences in the nuclei can be shown to have an influence even in early stages of development (e.g. in the merogons or hybrids in frogs, p. 358), but they are certainly not all-important. Thus for embryology the cytoplasm is as fundamental as the genes.

Equally, of course, one must not forget that an egg will not develop into even the general framework of an animal unless it is provided with nuclei. There can be no doubt that differentiation results from the interaction of the division-products of the original zygotic nucleus with the already-present ooplasmic regions of the egg. Neither cytoplasm nor nucleus can be disregarded: in fact the most important subject to discuss is how they affect each other.

We can, then, put into the following form the knowledge with which we have to approach the problem of how development is brought about:

(1) There are local differences with the cytoplasm of the newly fertilised egg. The nuclei, with the genes contained in them, react with the cytoplasm with which they are in contact; and the interacting system of nucleus and cytoplasm may also be affected by substances diffusing from neighbouring regions, as in the process of induction.

(2) As a result of the nucleus-cytoplasm reactions there are formed, firstly new duplicate genes (which allow for the multiplication of nuclei), and also substances of some kind which pass into the cytoplasm and modify Tits

(3) These ‘immediate gene products’ may interact with each other in the cytoplasm, or with other substances which are already there. There is likely to be quite a complicated series of reactions of this sort before the appearance of the differentiated cytoplasmic constituents which characterise the various fully developed tissues of the adult animal. As these reactions go on, the cytoplasmic environment of the nucleus will be altered, and this will affect the nature of the nucleus-cytoplasmic reactions. The gradually changing constitution of the cytoplasm may also be expected to alter the nature or intensity of the activities of the immediate gene products. We must therefore be prepared to find ourselves faced with a double cyclic system, of the kind pictured in Fig. 16.1.

The double cycle of intra-cellular reactions. The genes in the nucleus acting on cytoplasmic substrates, both reproduce themselves and control the formation of ‘immediate gene products’, a, b, c, etc. These then use cytoplasmic raw materials (i) perhaps to reproduce themselves identically, in the manner of plasmagenes, and (ii) to elaborate the final cytoplasmic constituents, P, Q, Retc., which are the substrates or raw materials which condition the activity of both the genes and the gene products. (From Waddington)


(4) The whole system is organised in such a way that development tends to proceed along one or other of a number of alternative canalised paths. That is to say, the developmental reactions tend to go towards one definite type of end-result or another; and while development is going on, the system has some power of self-regulation, so that no effect is produced by minor abnormalities such as slight losses of material or the substitution of one recessive gene for a dominant one.

It is now necessary to discuss in rather more detail the successive phases in this system of reactions. We shall leave till a later chapter the questions relating to morphogenesis in the strict sense, that is to say, the moulding of the developing tissues into definite shapes. Here we shall be concerned with the processes of chemical change during differentiation. This is one of the most active, important and controversial fields of biology at the present time. There are many different theories which will need to be considered; and since the problems get so far down to the common root of all biological phenomena, light may be thrown on them from very many different angles. We shall have to consider factual material drawn not only from experimental embryology of the old-fashioned kind, but from biochemical studies and from the genetics of microorganisms as well as higher forms. If is often difficult to assess the relative importance of these various types of evidence. On the one side we may have what seems very precise and definite biochemical information, expressed in terms of nucleic acids, enzymes, phosphate bonds and so on— but we have to ask ourselves just what is the connection between this and the phenomena of development which we are trying to explain, and consider whether it really is more enlightening than theories which operate with less clear-cut concepts (ranging all the way from genes to organisers and ooplasms) which are further from chemistry but nearer to the embryos.

The fourth point mentioned above may be dealt with first. It is a platitude, but an important one, that the body of a multicellular animal is made up of tissues which are rather distinct from one another. Even when changes in the normal course of tissue differentiation are brought about (e.g. by induction, or the action of lithium on the amphibian mesoderm, etc.) the altered tissue is usually switched from one into another of the well recognisable types—from epidermis to neural tissue, or chorda to somite. Intermediate types occur rarely, and when they do (as for instance the ‘palisade tissue’ found as a ‘weak’ reaction to a neural-inducing stimulus) they often later develop into something more normal, such as ganglion tissue in the case of palisades. The developmental reactions, therefore, tend to follow one or another of a number of definite paths, which lead to rather well defined and distinct end-states. Further, there is abundant evidence from all the regulatory phenomena which are so common during development, that even if the conditions in a developing tissue are made somewhat abnormal, the epigenctic system is often able to compensate for this, so that the normal end-state is nevertheless attained. Both these points can be expressed visually by means of a diagram such as that of Fig. 16.2, which represents what has been called the ‘epigenetic landscape’ (Waddington 1940a, q.v. for further discussion).

1. The effects of cytoplasm on the nucleus

In earlier chapters we have seen several instances in which localised areas of cytoplasm must have exerted an effect on the nuclei which move into them. For example, when any cleavage nucleus comes into the formation centre of the Platycnemis egg, a reaction takes place and a substance is produced which diffuses forwards to the differentiation centre (p. 126). Again any nucleus which reaches the grey crescent region of the amphibian egg can take part in the development of the organisation centre. And all the mosaic eggs similarly show that the role which a nucleus plays in development depends on the type of cytoplasm in which it lies. Rather little is known about the nature of the effects which cytoplasm has on nuclei. There are a few well-known cases in which the reactions produce alterations of the nucleus which are easily visible in conventional microscopical preparations (Review: Mather 1948a). One of the best known of these occurs in the eggs of Ascaris, a nematode parasite in the gut of the horse. This possesses only one pair of large chromosomes (polyploid races with two or four pairs are also known). These chromosomes are not very typical ones; in place of the normal single centromere or ‘spindle fibre attachment’ they are provided with a whole series of them extending along the centre section of each long chromosome. A visible nuclear differentiation takes place very early, since from the twocell stage onwards the chromosomes in most cells break up into small fragments, each provided with only one centromere, and the distal centromere-less ends (which are heterochromatic) are thrown out of the nucleus into the cytoplasm. Only in that lineage of cells which eventually gives rise to the germ cells do the long chromosomes retain their original configuration (Fig. 16.3). Some earlier authors (e.g. Zur Strassen) suggested that the phenomenon was due to factors residing in the chromosomes themselves, supposing that at each cleavage mitosis there was an unequal and orientated division by which a specially coherent chromosome was segregated into this lineage of cells. But Boveri, who was the first to describe this process of ‘chromosome diminution’, showed by a study of abnormal cleavages in dispermic and centrifuged eggs that the retention of the original structure is dependent on the type of cytoplasm into which the nucleus moves (discussion in Schleip 1929). The type of cytoplasm in which the chromosomes remain coherent can, it is claimed, be recognised not only by its location in the egg but by the presence of fewer vacuoles than there are in the rest of the egg (Bonoure 1939).


The ‘epigenetic landscape.’ A symbolic representation of the developmental potentialities of a genotype in terms of a surface, sloping towards the obser ver, down which there run balls each of which has a bias corresponding to the particular initial conditions in some part of the newly fertilised egg. The sloping surface is grooved, and the balls will run into one or other of these channels, finishing at a point corresponding to some typical organ. (From Waddington 19546.)

Very similar examples of a rapid differentiation of nuclei following a single division can be seen in a few other animals and in the pollen grain formation of some plants.

In certain animals, differences can be seen between the nuclei in different tissues. It is indeed usual for the nuclei to differ somewhat in size, general intensity of staining and perhaps in the number or size of nucleoli, but the significance of these characters is obscure. A more definite type of difference, found in certain groups, is the formation of polyploid nuclei, containing multiples of the normal diploid number of chromosomes. This is rather common in insects (Reviews: White 1954, Geitler 1948). In many tissues the chromosomes divide, and something rather like a process of mitosis may occur in an abbreviated form without any accompanying division of the cell body or of the nucleus. The resulting chromosome threads may lie in a loosely packed mass, or each may adhere closely to its partners. The latter process gives rise to configurations such as those which are best seen in the cells of the salivary glands of dipteran larvae, but which occur in a less perfectly developed form also in the Malpighian tubules, the linings of the gut and elsewhere. In nuclei with such “polytene’ chromosomes, the genes must be represented many times over, and different degrees of polyploidy seem to characterise the different tissues. It is theoretically possible that the relation between the quantity of gene and its effectiveness is not the same for all genes, so that a multiplication of the whole set of chromosomes would alter the effective balance between the genes. The common occurrence of polyploidy in some form or other in differentiated insect tissues does perhaps suggest that it is related to the varying activity of the nuclei in the different tissues and a similar suggestion has been made, chiefly with respect to plant material, by Huskins (1947) and Huskins and Steinitz (1948). However it must be admitted that most animals and plants which are wholly polyploid, and start life with an abnormal number of chromosome sets in the fertilised egg, do succeed in developing very normally and show little sign that the effective balance of their genes has been altered. Moreover, Staiger and Gloor (1952) have described a lethal factor (Ip!) in Drosophila hydei which has a colchicine-like effect on the mitoses in the cells of the larva, and thus leads to the formation of highly polyploid cells (up to 28-ploid). A similar effect can be produced by cold shock treatment (Gloor 1951). The damaged cells in most cases eventually die, but there is evidence that brain cells, for instance, may retain their differentiation and function as normal constituents of the brain even when their chromosome number is considerably larger than usual; and there is no indication that as the chromosome number becomes altered they tend to assume some other histological type. This makes it difficult to believe that the differentiation of tissues in insects is directly related to ploidy.


Cleavage in Ascaris: a, beginning of the second cleavage, b; later stage of the same cleavage, with the chromosomes becoming fragmented, and their ends (e) lost in the cytoplasm, in the anterior AB cells; ¢, 4-cell stage from animal pole; d later stage of 4-cell stage from the side, when cell P2 has moved round; chromosome fragmentation and loss of ends is occurring in cell EMSt. (After Boveri.)

A remarkable example of nuclear differentiation has been described by Lindahl (1953) in the echinoderms, in which the micromeres at the vegetative end of the egg become haploid;* in this case there is no evidence to what extent this is a cause or a consequence of the differentiation of that region of the embryo. Green (1953) finds similarly that the mesenchyme of the tail tip in anuran tadpoles is haploid.

A very peculiar situation appears to occur in mammals, in which there is evidence that the chromosome number may vary from cell to cell in the same tissue. In insects such as Drosophila, on the other hand, the loss of one of the chromosomes from a cell always has a definite effect on its development. Perhaps the apparent ineffectiveness of abnormal chromosome numbers in mammals depends on an ability of substances to diffuse from cell to cell, so that the gene-balance within a large mass of tissue is more important than that within individual cells; or perhaps the abnormalities arise too late in development to have much influence. The question requires much further study (see Beatty 1954).

There is also some evidence of a more biochemical nature which indicates a differentiation between the nuclei in various tissues (cf. Brachet 1952a, b). Thus in Amphibia, Brachet has shown that the nitroprusside reaction (indication of -SH groups) is positive in all the nuclei of the morula, but almost disappears from the ectodermal nuclei of the neurula, although it still remains strong in the neural tissue and in the notochord and mesoderm. Differences in the nuclei of the various regions of the amphibian gastrula in the incorporation of amino-acids are described on p. 204. Dounce (1954) has investigated the enzymes contained in nuclei isolated from various adult tissues, and although the techniques of isolation and handling of nuclei are not at present absolutely satisfactory, he finds strong evidence that the enzymatic properties differ considerably between nuclei. On the whole the nuclei in a tissue are rich in just those enzymes which are also found in the cytoplasm of the cells and it appears that this cannot be due solely to the contamination of the preparations of nuclei. Again Marshak (1951) claims that the ribose nucleic acid (but not the desoxyribose nucleic acid) of nuclei is very different in chemical constitution in the various tissues.

1 This has been denied by Makino and Alfort, 1954, Exper. 10,

2. Effects on chromosomes and genes

The formation in insect tissues of polytene nuclei, containing such large chromosomes as we see in the salivary glands, makes it possible to inquire whether the fine-grain patterns of the chromosomes are similar in different tissues. A well-formed polytene chromosome consists of a large number of threads lying side by side, each thread consisting of an alternation of refractive and deeply staining segments with less deeply staining stretches; the former contain much desoxyribose nucleic acid, the latter much less. By cytogenetic methods, the position of a considerable number of individual genes has been determined very closely; in fact it may be possible to show that a gene must be located within a certain particular deeply staining segment or band. If the activity of the genes is different in different tissues, it might be that the appearance of the bands would show some signs of this. The investigation of this point is complicated by the fact that the classical type of ‘salivary gland chromosome’ is the end-product of a long course of differentiation, and in the other tissues of Drosophila the process does not usually go so far but stops at an early stage in the sequence. Kosswig and Shengun (1947) were deceived by this into the conclusion that the detailed structure of the chromosomes is very different in salivary glands, Malpighian tubules, gut, etc. Slizynski (1950) showed, however, that all the major landmarks of the chromosomes can be recognised in all tissues in which there is a reasonably good development of polytene chromosomes, so that any differentiation of the chromosome banding must be on a rather minute scale.

Several workers, such as Beerman and Mechelke in Bauer’s laboratory in Germany and Pavan in Brazil, have recently discovered species in which the polytene chromosomes are well enough developed in a variety of tissues for their structure to be studied in detail. They have found clear evidence that individual bands, which may correspond to single genes, may be differently developed according to the tissue in which they lie; moreover some bands can be shown to pass through characteristic cycles of change, which appear to indicate the occurrence of important metabolic events at particular times in development. This is critically important evidence for the supposition, which has always seemed reasonable on general grounds, that the degree of activity of a gene depends on the cytoplasm surrounding it, and varies not only from tissue to tissue, but with the epigenetic situation within any one tissue.

Beerman (1952) has recently made a detailed study of the polytene chromosomes of several tissues of the species Chironomus tentans in which they are very well developed. He finds that there are characteristic differences in the general appearance of the chromosomies in the tissues studied; in the salivary gland they are rather compact cylinders, in the Malpighian tubules and rectum they are peculiarly kinked, and in the midgut they have the form of spirally wound flat strips. In all tissues, however, the same sequence of bands can be recognised, so that in all cases the complement of genes appears to be complete, as far as the cytological evidence goes. It is very important to observe that individual bands show characteristic appearances in the different tissues. This can be seen in Fig. 16.4, which shows a section of chromosome which can be identified with certainty by the presence of a small inverted section in which no pairing occurs. It can be seen, for example, that bands 1 and 2 appear swollen and ‘puffy’ in the rectum, but compact in the other tissues, while band 3 is puffy in the salivary glands and Malpighians, band 4 again in the rectum, and so on. Comparable differences may be observed when one compares the chromosomes of the same tissue at different stages of development, for instance at the larval and pupal periods. This is very convincing evidence that the state of activity of a band varies according to the tissue. Beerman suggests that the swollen and puffy appearance indicates a high metabolic activity, and he showed in fact that if larvae are brought out of the cold, in which their metabolism has been reduced, into a higher temperature, there is a rapid formation of droplets, visible by phasecontrast, in the neighbourhood of the most highly developed ‘puffs’. Mechelke’s work (1953) was done on another chironomid, Acricotopus lucidus in which the salivary gland is subdivided into three lobes, a forelobe, mainlobe and sidelobe. All of these contain well-developed polytene chromosomes. In general the banding is rather similar in all three lobes, but there are one or two very clear-cut cases of differential activity, one of which is illustrated in Fig. 16.5. In the mid-larval stages, region no. 33 of Chromosome 1 in the forelobe is enormously swollen into a fan-shaped mass (a so-called ‘“Balbiani ring’); in the other two lobes the same region has a perfectly normal structure. The swelling in the chromosomes of the forelobe begins to retract at the end of larval life and during the prepupal stage, exactly at the time when this lobe produces a brownish secretion. In Pavan’s case (Pavan 1955) there is also a transitory swelling of particular bands at particular times in certain tissues but not in others. This seems as direct evidence as one could hope for of the activity of individual genes at characteristic times and places.


A certain region in the third chromosome of Chironomus tentans, identifiable by a simall inversion which causes a failure of pairing: 4 is from a cell in the mid-gut; b, salivary gland; c, Malpighian tubule; d, rectum. Note that the bands (or group of bands) 1-6 are identifiable in every case, but may be differently developed in different tissues. (After Beerman 1952.)


Figure a, the normal structure of part of Chromosome I of Acricotopus lucidus from the main lobe of the salivary gland; b, the ‘Balbiani ring’ developed from region 33 in the forelobe of the larva; c, a regressing Bal biani ring from the forelobe of a prepupa; d, a fully regressed Balbiani ring from a later stage. (From Mechelke 1953.)

The fact that a given gene produces a different intensity of effect in different types of cytoplasm is, of course, obvious enough from the mere occurrence of the differentiation of gene-controlled processes. It is also demonstrated very clearly in certain particular cases. Baltzer (1940, 19520, c) and his students have carried out many experiments in which an egg of one species of urodele has been fertilised by sperm of some other species e.g. Triton taeniatus or palmatus egg fertilised by T. cristatus sperm. These hybrids are often viable, but some combinations eventually die, usually at fairly late stages of development. A more interesting situation is produced if, after the fertilisation but before the conjugation of the nuclei, the egg nucleus is sucked out with a pipette. This leaves only the foreign sperm nucleus in the cytoplasm of the other species; such animals are known as hybrid merogons. In all the combinations tested, they die before completing development and at an earlier stage than do the corresponding hybrids in which the female nucleus has been left in situ. Presumably the origin of the nucleus and cytoplasm in the hybrid merogons from different species makes it impossible for them to interact in a satisfactory manner. In some combinations, e.g. the Triton 9 + Salamander $ hybrid (with female nucleus intact), the lethality affects all cells of the embryo more or less equally. In others, however, some tissues may be much more strongly affected than others. In the hybrid merogon in which there is only a T. cristatus nucleus in taeniatus cytoplasm, it is the head mesoderm which suffers most severely and which dies earliest. This must mean that there is some reaction between the nucleus and cytoplasm of this particular tissue which cannot be properly performed when they belong to different species (Fig. 16.6).

It is noteworthy that in several such cases the merogonic tissue which would die or fail to develop if left in situ can be kept alive for a considerable time, and will often continue its differentiation, if it is grafted into a normal host embryo (e.g. Hadorn 1937). Apparently the substances which the merogonic tissues cannot make are diffusible and can reach it from healthy tissue in the neighbourhood. Their nature is quite unknown, and would seem likely to repay investigation. THE ACTIVATION OF GENES BY THE CYTOPLASM 359

A very remarkable example of cytoplasmic activation of genes, and one very hopeful for future investigation, occurs in the protozoan Paramecium. In this animal, there may be found one or other of a series of antigens, which can stimulate the formation of corresponding specific antibodies when injected into rabbits. Paramecium has one great advantage as an experimental animal in that it is possible to arrange for a nucleus of one genetic constitution to get into cytoplasm which has been formed under the control of a nucleus of a different type or under the influence of differ


, HOMER AW sm Tk A x




1. An egg of Triturus palmatus when fertilised by sperm of T. cristatus develops normally.

2. If the palmatus egg nucleus is removed before fertilisation, we obtain a ‘hybrid merogon’, with a haploid cristatus nucleus in palmatus cytoplasm; this dies at the early neurula stage.

3. The hybrid merogon combination of palmatus cytoplasm with alpestris nucleus dies rather later, while (4) the reciprocal combination of palmatus nucleus in alpestris cytoplasm dies earlier. On the right is a diagtammatic longitudinal section through a hybrid merogon of cristatus nucleus in taeniatus cytoplasm (which behaves like the cristatus in palmatus combination shown in Figure 2). The dots show the region in which the mesoderm

cells become necrotic. (From Baltzer 1940.)

ent environments. This may occur during the process of conjugation, in which two Paramecia, possibly of different genetic constitution, come together and each inseminate each other. Very little if any cytoplasm is normally exchanged during this process (though it is possible to arrange for this to happen if the experiment requires it), which therefore gives rise to two individuals, each of which contains a similar hybrid nucleus, but each with its original characteristic cytoplasm.

Experiments of this kind (Sonneborn and Beale, 1949, Beale 1952, 1954) have shown that the immediate control of antigen formation is due


to some factor in the cytoplasm. For instance, when conjugation occurs between two Paramecia, one carrying the antigen A and the other B, the two individuals which separate again will be alike in their complements of genes, but unless some interchange of cytoplasm has occurred, each continues to produce its characteristic type of antigen. However, the responsibility of the cytoplasm for the antigens is not absolute; the genes determine the range of possible antigens which the Paramecium can form. So far three main gene-loci have been studied, known as D, Gand S. Each strain possesses its characteristic alleles at each of these loci. Normally, only one locus is active at a time. The important point for our present discussion is that it is the condition of the cytoplasm which determines which of the loci shall be in operation (Fig. 16.7). Exactly what is implied

(a) g°° (s) ® (60 Parents eG) TO @ -----¢ e )

F, animals (4) (3s) ___ o*) 8 (s)

finally at 25° (6) g (5) (4) g® (s) { |

F, animals (4%) g(s)_ ____ 4 (g” s)

after 5 fissions (4%) * (5) > (g s)

F; animals d% (99 5)

Oe (gs) g” g ) finally at 29° d (g* s) 6 acs (3 5 FIGURE 16.7

The results of a cross between Paramecia of stocks 90 and 60. The former have been kept at 25° C., and the state of their cytoplasm activates their genes controlling G antigens (i.e. g9°); while the latter have been at 29° C. and D antigens are being produced. In the first few generations of the F,, the G or D states of the cytoplasm persist, and bring into action the corresponding g or d genes in the hybrid: thus the individual with shaded cytoplasm now forms g®° and g® antigens, and that with unshaded cytoplasm d°° and d®, After some time, the cytoplasm of later generations adjusts itself to the temperature at which the stocks are kept; and when it does so, activates the corresponding genes. (From Beale 1954.) THE ACTIVATION OF GENES BY THE CYTOPLASM 361

by ‘the condition of the cytoplasm’ is still obscure; but, whatever it is, it can be altered by a number of agents, for instance by the temperature at which the strain is being cultured, the amount of food available, the osmotic pressure of the medium, etc. This would seem to open the possibility of discovering how the activation is brought about and the nature of the cytoplasmic properties on which it depends. This is a clear-cut example of the activation, by different types of cytoplasm, of different specifically corresponding genes; and the fact that this occurs, not in different parts of a single body but in the various members of a strain of unicellular organisms does not make the phenomenon any the less relevant to the normal processes of development.

3- Complete or partial inactivation of genes?

There is therefore a fairly solid, and increasing, body of evidence which indicates that the nuclei of differentiated tissues become influenced by the cytoplasm with which they are associated. It remains to discuss how far this influence extends. Are we to imagine that certain genes become completely inactivated or even lost; or is it more likely that we are dealing with a merely quantitative speeding up or slowing down of the geneactions?

There is rather little direct evidence on the matter from the side of embryology. It is true that in many plants almost any part of the organism can be caused to produce a whole plant and must therefore contain the whole set of genes. But plant tissues are not so highly differentiated as those of animals, and one can hardly adduce the evidence of their powers of regeneration to prove that animal cells also always contain a full set of genes. Among animals, it is difficult to find clear-cut cases in which cells can be conclusively proved to have first differentiated in one way and later to have shown a capacity to change into some other type which might be supposed to demand the activity of previously unused genes. When differentiated vertebrate cells are grown in tissue culture (Review: Willmer 1954), they ‘modulate’ into less-specialised forms which may appear to be dedifferentiated, but they do not re-acquire the ability to develop into some tissue other than the one from which they were originally derived. However, there are fairly convincing cases of such a ‘metaplasia’ (i.e. a renewal of developmental plasticity) in the ascidians, the embryos of which are highly ‘mosaic’ at a rather early stage while the adult cells exhibit considerable flexibility during the processes of regeneration and budding (Harrison 1933). We have also seen (Chapter XIV) that there is good evidence for some degree of metaplasia during vertebrate regeneration. 362 PRINCIPLES OF EMBRYOLOGY

There is a considerable body of evidence from genetics which, although it by no means settles the question, tends to suggest that all genes normally remain in being in all types of differentiated cells.

It is, of course, common to find that a given mutant gene produces a rather localised abnormality and appears to be inactive elsewhere, and if this evidence were taken at its face value, there would be nothing to prevent our supposing that the gene had been completely inactivated or lost in those regions in which it has no visible effect. However, Waddington (1953) has shown that several genes in Drosophila are actually in operation in regions in which their influence is not obvious at first sight. For example the well-known gene vestigial causes a severe reduction in the size of the wings, but seems to have no influence on the immediately neighbouring thorax. But in flies which are homozygous both for vestigial and a gene such as dachsous, which does affect the thorax, it can clearly be seen that vestigial is active not only in the wings but also in the body (Fig. 16.8). One must assume that the effect of the vestigial gene on the thorax normally falls below some threshold and produces no visibly abnormal result unless the development of the thorax has already been upset, and its canalisation weakened, by the action of some other mutant such as dachsous. There are certainly many other cases of such subthreshold effects, as would be expected if all genes are effective, to a


(On the left). The gene dachsous causes a slight enlargement of the thorax of Drosophila; vestigial reduces the wings to vestiges, and apterous has a still more severe effect of a similar kind. In the fly shown, which is homozygous for all three genes, it is clear that vestigial and/or apterous have sub-threshold effects on the thorax which become effective in the combination with dachsous. (From Waddington 1953.) (On the right.) Sections through the spermathecae of various mutants, showing how the shape is affected by genes whose main action is elsewhere. (From Dobzhansky 1927.) THE ACTIVATION OF GENES BY THE CYTOPLASM 363

greater or lesser extent, in all tissues, though with different intensities in different parts.

A similar conclusion is suggested by the fact that careful metrical study often reveals an activity of a gene in a tissue which it had previously been thought to leave uninfluenced. Thus Dobzhansky and Holz (1943) induced a number of mutations, affecting the eye colour, bristles or other external characters, in long inbred strains of Drosophila melanogaster. Each mutant strain thus differed from the race from which it was derived only by the actual mutated gene. By comparing two corresponding races it could be shown that nearly every mutant produced alterations in an apparently quite unrelated character (the shape of the spermathcca) as well as in the eye colour, etc. by which it had originally been detected. It is difficult to suppose that this was merely fortuitous and it seems much more probable that the evidence can be accepted as indicating that all, or nearly all, genes are active in every tissue (Fig. 16.8).

Another piece of evidence tending in the same direction is given by some work of Demerec (1934, 1936) and depends on the phenomenon of somatic crossing over. If a Drosophila is heterozygous for two linked genes, such as singed (bristles) and yellow (body colour), during development a process of crossing over will, in a few cells, take place at a mitotic division, so that one of the daughter-cells becomes homozygous for singed and the other for yellow, These cells will each give rise to a small patch of tissue, and if this forms part of the body surface, one will see small twin spots, a yellow-coloured one and one with singed hairs. Demerec bred flies which were heterozygous not only for yellow and singed but also for one of a number of small deficiencies located in the same chromosome. He found that in most, but not quite all, cases the somatic crossing over now gave rise only to a single spot, either a yellow one or a singed one, the other partner spot being missing. This was interpreted to mean that the daughter-cell which had become homozygous for the deficiency (and therefore lacked entirely the genes involved in it) were not able to survive: the deficiency in fact was operating as a ‘celllethal’. If this is true, it means that all the genes involved in the cell-lethal deficiencies are not only normally operating in the hypodermis cells, but are active in such an important way that in their absence the cell dies. Since the deficiencies were selected at random, and were not known previously to contain genes active in the hypodermis, this is rather good evidence that all genes are active in these cells, most in fact being essential to life. And presumably the same conclusion must apply to all the tissues in the body, although the relative importance of the various genes could not be expected to be always the same. 364 PRINCIPLES OF EMBRYOLOGY

There are therefore some grounds for thinking that all genes may be active, and producing effects of some kind, in all the cells of the body, even in those in which, owing to the canalisation of developmental processes, the influences of the mutant alleles do not suffice to produce any divergence from the normal. It must be emphasised that this suggestion remains no more than a hypothesis, and that it is quite possible that in some tissues certain genes become completely and irreversibly inactivated or lost: the point will not be finally decided until it is possible to transplant nuclei from differentiated cells into cytoplasms of an earlier and not-yet-determined stage and to discover whether such nuclei still retain the full range of developmental potentialities. Briggs and King (1952, 1953) were the first to achieve any important success in this. Frogs’ eggs were parthenogenetically activated by pricking with a glass needle, and the egg nucleus removed (fortunately it can be located with some certainty in this form). A nucleus with a little associated cytoplasm from a cell of a later embryo was then injected into the enucleated egg. Cleavage followed in a fair number of cases. With nuclei taken from morula, blastula or early gastrula stages, complete development of the host egg into a fully differentiated larva sometimes occurred. This proves that, as might be expected, no irreversible change has occurred to the nucleus during these early stages, before the onset of determination, let alone of cellular differentiation.

Mote recently, similar results have been reported with nuclei from the determined but not yet differentiated tissues of the late gastrula. Waddington and Pantelouris (1953), working with newts’ eggs, found that such nuclei were just as good as earlier ones for enabling cleavage to occur, but in their material the host eggs always stopped at the beginning of gastrulation, whatever the age of the transplanted nucleus; this was probably due to the inadequacy of the technique rather than of the nuclei. King and Briggs (1954), however, have succeeded in transplanting such nuclei in frogs, and have shown that they can control the development of completely normal tadpoles. Thus determination does not involve any irreversible loss of gene function; it still remains uncertain how far this is also true of differentiation.

4. The mechanism of gene activation and inhibition

One would, of course, like to understand the mechanism by which the cytoplasm influences the nucleus and stimulates or inhibits the activity of various genes. There are several possibilities which must be envisaged. In the first place, so long as division continues in a cell-lineage, the genes in the nucleus must be synthesising duplicates of themselves so as to provide the increasing number of chromosomes; and at the same time and probably even after all division has ceased, the genes must manufacture substances which pass into and influence the cytoplasm. We shall discuss the nature of these substances in the next section. The point which is being made here is that the genes are producing substances; and in order to do this they must use some raw materials. It is therefore possible that one of the factors which controls the intensity with which the genes operate is the availability of the relevant raw materials.

One form which the competition for raw materials might take would be that the actual total quantities of certain substances set a limit to the activity of particular genes which specially required them. It is perhaps rather improbable that competition of this kind plays an important part in development. When an egg is cut into fragments the amounts of cytoplasmic raw materials available to the nucleus will of course be reduced. Nevertheless, if the cut is made in the right direction a normal embryo may be produced. It seems more plausible to suggest that it may be not the total amounts of substances but rather their relative concentrations which influence the activities of the genes.

There are, of course, other theoretically possible types of control mechanism; for instance some regions of cytoplasm may contain substances which specifically stimulate or inhibit particular genes, somewhat in the manner of co-enzymes or enzyme inhibitors: or the products which the gene passes into the cytoplasm may themselves tend to increase or diminish further gene activity. Our knowledge is so slight that it is hardly profitable to enumerate any more possibilities. It is worth pointing out, however, that it is perhaps at this level that one should look for the explanation of the fact that in any given species of animal there are only a limited number of rather sharply distinct alternative paths of development. We shall show later that this is an expected consequence of any system in which a number of different chemical processes either compete with another for a limited set of substrates, or interfere by other kinds of mutual stimulation or inhibition. We shall see reason to suppose that the cell contains another set of similarly competing or interfering reactions, namely those which operate between the products which the genes pour out into the cytoplasm. It is not clear to which of these two systems—that involving genes and their raw materials, or that involving gene products and their raw materials—the formation of alternative pathways of development is due, but we shall discuss the matter more thoroughly in connection with the gene-product system, which will be dealt with in the next section.

Our information about the mechanism of the cytoplasmic control of gene activity is, perhaps, fullest in connection with the phenomenon of embryonic evocation; but even so, it is not enough to carry our theories on to firm ground. When a group of embryonic cells is induced to enter on some particular path of differentiation, say towards the formation of neural tissue, we know that the detailed character of the tissues produced will be determined by the specific nature of the reacting cells. In a few cases we can be certain that the specific character of the competent cells is an expression of the genes they contain; for instance when melanophores are induced in genetically white or black axolotl tissue. And it is most probable that in other cases, in which no analysis by genetic methods has yet been made, most or all of the specific differences involved are to be attributed to genes rather than to cytoplasmic factors. We have then good grounds for supposing that when gastrula ectoderm is evocated to form neural tissue, the set of gene-activities stimulated within it are different from those which would be involved in the development of epidermis. We have already described the basic information about the nature of the evocation process (p. 206), but it needs to be examined again from the point of view of gene activation. However, some of the theories about it also involve the products, as well as the precursors, of gene activity, and this discussion also will be postponed till chapter XIX.

Suggested Reading

Baltzer 19526, Beale 1954, pp. 77-123, 148-163, Beerman 1952, King and Briggs 1953, Mather 19484.

   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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