Talk:Waddington1956 17

From Embryology


IN CONSIDERING separately the influence of the cytoplasm on the genes, by way of activation and inhibition, which was dealt with in the last chapter, and the influence of the genes on the cytoplasm by the production of substances active in development, we have made a distinction which, however convenient, is to some extent artificial. It will be shown later that in all probability the substances produced by genes at an early stage of development are themselves capable of affecting the levels of gene activity at later stages. The gene-cytoplasm complex is a single system, between the parts of which reciprocal interactions occur. But it is easier to discuss the individual steps in such circular reactions separately at first, and to try to put them together again later.

1. The parts of the cell

Information about the production of substances by genes comes partly from genetics, but very largely from cytological, embryological and biochemical studies. It will therefore be advisable to begin this chapter by a summary description of the structure of a typical embryonic cell, mentioning the cell-parts which are most important for the subsequent discussion (Fig. 17.1).

The cell nucleus consists of the chromosomes, nucleoli and nuclear sap, all contained in a nuclear membrane. In the chromosomes one can roughly distinguish two types of material (see White 1954): the euchromatin, which shows the typical staining behaviour from which the chromosomes (‘the coloured bodies’) derive their name; and the heterochromatin, of which there may be more than one kind, whose staining behaviour diverges in various ways. The staining reactions of the euchromatin are in the main due to their content of desoxyribonucleic acid (often abbreviated to DNA) which can be more or less specifically recognised by the Feulgen reagent and less certainly by many other stains. This substance becomes condensed on to the chromosomes at the time of cell division, but it may spread more diffusely throughout the nucleus in interphase. The different staining behaviour of heterochromatin is the result in part of the fact that the phases of the nucleic acid cycle in it are not synchronised with those of the euchromatin. It is probably also due in part to a greater concentration in the former of the other type of nucleic


acid (ribose nucleic acid, or RNA). The chromosomes also contain protein. Much of this is in the form of protamines and histones, two rather peculiar types of proteins which are characteristic of chromosomes and hardly known elsewhere. There are undoubtedly also other types of proteins in chromosomes, but there is as yet little agreement about their nature (cf. Mirsky 1952).


Diagram of the main elements in cell structure. Inside the nuclear membrane (Nim) are pairs of chromosomes; each chromosome may contain not only euchromatin, which stains deeply at mitosis, but also ‘heterochromatin’, possibly of more than one kind (/,, hy); each also possesses a centromere, or spindle attachment (a) and some chromosomes form nucleoli (N/) at specific places along their length. In the cytoplasm, there may be a special granular or vesicular region, the ‘Golgi apparatius’ (G). Most of the cytoplasm appears clear, but it contains largish particles, known as mitochondria (M4, M,) and probably very small ultra-microscopic particles known as microsomes

(tty, Hg).

Attached to the chromosomes there may be one or more nucleoli. Typically each nucleolus is formed at one definite place on a particular chromosome, a so-called ‘nucleolar organiser’. The number of places which are active in this way probably varies in different tissues of the same animal; for instance very many nucleoli may be formed at a number of different places on the chromosomes of the amphibian oocyte nuclei, THE SYNTHESIS OF NEW SUBSTANCES 369

while only one or two appear in most somatic cells. The nucleoli contain basic proteins and RNA, but little or no DNA.

The last constituent of the nucleus is the sap. Unfortunately little is known about this, although it must be very important since it presumably contains both the substrates out of which new chromosomes are built and also the immediate products of gene activity. The main constituents are undoubtedly proteins (cf. Brown, Callan and Leaf 1950) and there is no nucleic acid.

The whole nucleus is enclosed by a nuclear membrance, which seems, in several, and perhaps in all, cases to be a double structure. An outer lipoprotein layer, a few hundred A thick, has a porous structure, the pores having a diameter also of three or four hundred A; this is supported on a thinner (c. 150 A) layer which shows no obvious structure in the electron microscope and is probably composed of an elastin-like protein (Callan 1951), on the nuclei of amphibian oocytes; Bairati and Lehmann (1952) believes that in Amocba the porous layer lies inside the structureless one). There is some evidence that the nuclear membrane is freely permeable to proteins, which makes it easier to see how the genes can effectively control the functioning of the cytoplasm (Anderson 1953, Stern and Mirsky 1953).

The cytoplasm consists of a clear “ground substance’, in which granules of various kinds are suspended. With microscopes using visible light, it is difficult to obtain much further information about the former. Studies with the electron microscope in recent years have been revealing a variety of laminar or fibrillar structures, often taking the form of very thin double membranes (Fig. 17.2). It is not yet clear, perhaps, to what extent these structures are the results of the types of fixation used to prepare the material (mostly neutralised osmic fixatives, which reveal almost no structure in the nucleus); but it seems certain that there must be quite elaborate structures of some kind or other in the apparently clear cytoplasm. (Sjostrand and Hanzon 1954.)

Most recent discussions have emphasised the importance of the various kinds of particles suspended in the cytoplasm. The first category of these are globules of fat or lipo-protein yolk, which act as reserves of energyrich raw materials. A more active role is played by the mitochondria, bodies which are large enough to be easily visible in the microscope (c. 0-5-3 in diameter, and sometimes ten or more times as much in length). They contain protein, lipids, and a little RNA. They are usually thought to contain little DNA, but Chayen and Norris (1953) have recently shown that in actively metabolising interphase cells, much of this substance is located in cytoplasmic granules, from which it easily passes 370 PRINCIPLES OF EMBRYOLOGY

into the nuclei if the cells are damaged, or treated with inappropriate histological methods. The main physiological activity of the mitochondria seems to be the performance of co-ordinated sequences of respiratory enzymatic processes. Electron microscope studies show them to have quite an elaborate internal structure, which again consists largely of closely opposed double membranes (Palade 1952).


Diagram of structures seen in the cytoplasm of mouse pancreas cells (elec tron-microscope studies of ultra-thin sections). The circles to the right show

higher magnifications. The sausage-shaped bodies are mitochondria, which

have an internal structure consisting of double membranes. (From Sjéstrand and Hanzon 1954.)

The other main class of cytoplasmic granules generally found in cells is referred to by the term ‘microsomes’. They are typically at or just below the limit of visibility in the ordinary microscope (diameter usually 60-150 my) but can be sedimented out of the clear cytoplasm by ultra-centrifugation, They contain little lipid, and are mainly characterised by their richness i RNA. In addition they contain protein, but usually show little enzymatic activity, except that if the cell from which they are isolated is of a kind in which a specific enzyme is found (as trypsin in the pancreas or amylase in the salivary gland) then this enzyme may be demonstrated in THE SYNTHESIS OF NEW SUBSTANCES 371

the microsomes. We shall see that one of the main theories concerning the chemical processes of development attributes great importance to the microsomes in the synthesis of cell-specific proteins. Their condition in the undamaged or uncentrifuged cell is still uncertain, since they range down to a size which is too small to be observed in living material. It is possible that in life they do not exist as separate particles, but that the ‘microsomes’ found after high-speed centrifugation are really the products of the breakdown of the membranous structures of the clear cytoplasm which are seen in Fig. 17.2.

In embryonic cells there are often many other granules which do not fall quite clearly into the two classes of mitochondria and microsomes (cf. Holtfreter 1946). As has been pointed out (p. 41), several different kinds of granule may be built in to the cytoplasm of the developing oocyte, coming either from the nurse cells or the germinal vesicle or arising in situ. It seems probable that during the early stages of development some of these are being gradually transformed into the typical forms of mitochondria and microsomes. Before this transformation is complete, they are referred to either by special names specially invented to cover particular cases (e.g. ‘a-granules’ and similar phrases) or by general terms such as ‘lipochondria’, etc. It is possible, also, that the microsomes may gradually develop into mitochondia (Brachet 1952).

2. Arguing from the gene to the substance

There are two main methods of approach to the problem of the production of substances by genes; one which starts from the genes and attempts to link them up with substances which can be identified as their immediate products, the other which starts from substances which are known to be under genetical control and tries to delve behind their antecedents until it reaches the genes. We shall consider the former approach first; it has so far not proved very productive.

Logically our first step should be to define the gene. Nowadays it is not easy to do this in an unequivocal fashion. Originally, a Mendelian factor was an abstract entity invoked to explain the numerical relations among the offspring of crosses. Then it was discovered that the factors are carried by chromosomes and the term ‘gene’ was introduced to refer to the physical entity which constitutes a unit factor. The difficulty is to know how to distinguish one unit factor from another. There are several possible criteria. The best known is based on crossing-over. Two genes are considered different if crossing-over can take place between them. One difficulty with this definition is that certain regions of chromosome are known (e.g. the Y chromosome in Drosophilia or the central parts of 372 PRINCIPLES OF EMBRYOLOGY

certain chromosomes in Oenothera) in which no crossing-over occurs. Further, the definition involves us in proving a negative. Shall we be satisfied if no crossing-over occurs in one thousand individuals, in ten thousand, one million, or how many (cf. pseudo-alleles, p. 375)?

Another criterion could be based on the breakages induced in chromosomes by x-rays or mutagenic chemicals. This would suppose that breaks must occur between genes and not through them and that the breaks would therefore suffice to separate the individual genes from one another. Such a criterion is, however, not easy to use in practice. Thirdly, we might try to base a definition of the gene on its physiological action, a gene being the smallest element which behaves as a unit in the developmental activity of the chromosomes. We shall see, however, that the evidence shows that this definition conflicts with the others, since parts of the chromosome which behave as separate units from the point of view of crossing-over may nevertheless influence one another’s developmental activity. It may be possible to interpret these influences as secondary interactions of neighbouring genes, but still the situation makes it difficult to provide a perfectly simple and clear-cut definition.

If, however, one looks more closely at the material structure of the chromosome which one is talking about, the reasons for this difficulty become easier to understand. The chromosome consists largely of protein which, throughout most of the life-cycle of the cell, is combined with a greater or lesser amount of nucleic acid. Now the basic structure of both protein and nucleic acid consists of a linear arrangement of small units. More is known about the proteins. The basic element here is a polypeptide link, the sequence in which is



| R

These are arranged in series which may contain many hundred individual links. Within such series there is a hierarchy of periodicities of different scales. The amino-acids (R) attached to the polypeptide chain may, for instance, be arranged in a repeating pattern, the repeat unit covering a fairly small number of individual links. Then there may be rather larger units corresponding to the unit cells of protein crystals. The protein molecules, as they may exist in solution, are a larger unit again. Virus THE SYNTHESIS OF NEW SUBSTANCES 373

particles provide a model of repeat units of a still larger size. The smallest recognisable units in chromosome structure (the bands in salivary gland chromosomes) are still larger. One can represent such a sequence by a series of letters such asabcde’ fg hi’ jk’ mnoPQRSTU

V’ W’ X’. Here the individual letters represent the smallest repeat unit.

The groups which are plain, dashed or underlined represent the next largest and lower-case and capital letters represent a still longer periodicity. In a protein structure of this kind there are many types of units, and different ways of defining the gene may lead to different results. We might in some cases find that all the lower-case letters were acting as one unit, while in other circumstances it might be the dashed lower-case letters which behaved separately to the undashed ones. There might even be an overlap between genes, in some cases the lower-case and the capital letters behaving as two units, while in other processes it was the underlined letters which went together. The usual crossover gene is thought to contain some three hundred of the small polypeptide links so there is plenty of room for complications of this kind. The nucleic acid is also a linear structure, and it seems likely that in it too the order in which the constituent groups are repeated can determine structures at least as complex as those of the proteins (Davidson 1954, SEB Symposium 1947). It may well be, indeed, that the factor which operates as a gene is a certain arrangement of chemically reactive places, which may sometimes be incorporated in a length of protein molecule, at other times in a nuclear acid fibre and at other times in the combination of the two. For our present purpose of investigating the nature of the reactions in which genes participate, we may be content to keep in the back of our minds the ambiguities in the precise meaning of the word and to understand it as referring to some sort of small section of chromosome which is acting as a unit.

One line of investigation into the activities of genes has attempted to discover something of their chemical nature by a study of artificial mutation. It was shown by Muller in 1927 that penetrating ionising radiation increases the frequency with which genes mutate; and Auerbach and Robson in 1946 found that certain chemical substances have a similar effect. In spite of the enormous amount of work which has followed the lead of these pioncer investigations little has been discovered which is really pertinent to our present problem. It has become clear that genes can be brought into a condition of instability by various treatments, but the effective physical and chemical agents are of kinds which can be expected to affect a large range of different structures, so that the fact that they are active in stimulating mutations does not make it possible to draw 374 PRINCIPLES OF EMBRYOLOGY

conclusions about the gene which are precise enough to illuminate the nature of gene-activity (Reviews: Lea 1946, Muller 1947, Catcheside 1948, Auerbach 1952).

The induction of high rates of mutation has, however, provided us with an enormous mass of genetic variations which have thrown light on our problem from other angles. Irradiation by x-rays, for instance, frequently causes chromosomes to break and rejoin in abnormal ways. From the study of such chromosome rearrangements, the fundamentally important

oint has emerged that the behaviour of a gene may in some cases be influenced by its position in the chromosome. This is the so-called position effect. Several theories have been proposed to account for it (cf. Lewis 1950, Serra 1949). There are two main hypotheses, which seem at first sight to be of rather radically different nature. The first accepts the conventional idea of the gene as a distinct and individual particle and supposes either that neighbouring genes produce substances which can react together (Offerman 1935, Stern et al. 1946) or that they interfere with one another by competing for the same substrates (Waddington 19394); if the diffusion of these substances is slow, it becomes important whether the genes are close together or far apart. The second involves a more profound change in previous ideas. It suggests that gene-activity is not to be attributed to circumscribed particles, which could be considered as separate ‘beads along the chromosome thread’, but that the basic elements are short stretches of chromosome which are not sharply bounded off against each other, but rather shade into or overlap one another. A change in the order of the chromosome thread will in that case alter the character of the fundamental reactions carried out by it (cf. Goldschmidt 1938, 1946).

It is not easy at present to decide finally between these two theories; it is significant, for instance, that Pontecorvo (1950) was led by a variety of the first theory to postulate that genes which take part in a series of reactions in which only a few molecules are involved in each cell will tend to lie close together, like successive machine tools on a production line; a special attempt was made to find such genes in a mould, Aspergillus; several genes controlling the production of biotin were duly discovered, all located very close to one another; but by that time Pontecorvo was feeling tempted to interpret the phenomenon by the second theory rather than by the first, which had led him to predict it (Pontecorvo 1952a, b).

The study of multiple allelomorphs has recently produced evidence which seems to reinforce the theory which postulates less definitely defined genes. It has long been known that there may be many different mutant forms of the same ‘gene’, if one may use the old terminology for the moment without begging the question. At one time it was thought THE SYNTHESIS OF NEW SUBSTANCES 375

probable that the different alleles differed only quantitatively, in efficiency or even perhaps in quantity of substance (cf. Goldschmidt 1938). More recently, several cases have been described in which the differences must be qualitative. For instance, Stern and Schaeffer (1943) showed that there must be two aspects to the activity of the gene cubitus interruptus in Drosophila, an ability to combine with a substrate, and power of reacting with the combined substrate; and they demonstrated that in the various alleles of this locus these two properties vary independently, some alleles having a high combining ability and low reactivity, others the reverse. Similarly, Waddington and Clayton (1952) found that the various alleles of the gene aristopedia in Drosophila vary independently in their effectiveness in altering the legs and the antenna of the animal, and cannot be arranged in any single quantitative series.

Recent work has, however, gone much further than such demonstrations that mutation may produce qualitative, and not merely quantitative, changes in genes. In the last few years, an increasing number of cases have been found in which crossing-over takes place between two genetic factors which according to all other evidence would seem to be alleles of a single locus (Review: Lewis 1951). The existence of such ‘pseudo-alleles’ is beginning to appear so widespread that one is bound to suspect that all apparent alleles may really be of this nature, that is to say, that the quantitatively or qualitatively altered action of each type of mutated gene is correlated with the particular stretch of chromosome which has become changed. If this is so, we shall have to envisage the genetic unit of activity as something which is considerably larger than was previously thought, a point of view which has been strongly urged for some years by Goldschmidt (1938). It remains very difficult to form a picture of the chemical nature of such active units, which would be much larger than normal protein molecules and perhaps similar in size to some of the viruses.

There is another category of position effects which also suggests some rather specific conclusions about the action of genes. It is quite commonly found that when a chromosome is broken and rearranged in a way which brings the heterochromatin into an abnormal position, the functioning of the genes which are now near to it becomes unstable, so that in some cells the genes function with full activity, while in others they are more or less inhibited (cf. Lewis 1950). This gives rise to a variegated or mottled effect, the degree of mottling varying somewhat from tissue to tissue or even in different parts of the same organ. Since many different genes show the same kind of behaviour in such rearrangements, it seems that the heterochromatin must exert some general influence on the activity of most or all genes; the nature of its action remains obscure, but is probably connected


with the importance of RNA in protein synthesis (cf. Serra 1949, Schultz 1952).

The ‘inactivation’ of the genes in these ‘unstable’ chromosome rearrangements is probably to be understood as a mutation to an inactive allelomorph, since in certain cases at least it may occur in germinal tissue and then breed true in the inactivated form. It has been suggested by McClintock (1951) that differentiation might depend on the occurrence in the different tissues of gene-mutations controlled by some mechanism of this sort, involving an interaction of heterochromatic and euchromatic segments of the chromosomes. But the hypothesis appears rather farfetched. In the examples known at present the mutations occur in a disorderly fashion, giving rise to flecks and spots which have little relation to the main anatomical features of the organism. Moreover, to explain differentiation we should need not only the orderly mutation of one gene, but of the whole complex set of genes active in the tissues concerned.

Another mechanism which may be related to gene mutation should be mentioned. Some years ago Avery showed that the characters of certain strains of bacteria (Pneumococcus) can be transferred to other strains by cell-free extracts. The strain thus ‘transformed’ continues to multiply in its new form. The ‘transforming principles’ have been shown in some cases to be composed of DNA, apparently unmixed with compounds of any other type. A considerable amount of work has been done on the genetical analysis of this most interesting phenomenon (cf. EphrussiTaylor 1951) but the mechanism of the transforming action is still quite obscure: the principles may operate by inducing specific gene mutations or in some other way. There are obvious analogies between these bacterial transforming principles and embryonic evocators, but there is little means of deciding as yet whether these are merely formal parallels or whether there is in fact any important similarity between the two mechanisms. The occurrence of embryonic induction by unnatural evocators would at first make it seem unlikely that the two phenomena are closely related; but this argument would have little force if the unnatural evocators operate by setting free the normal evocator from an inactive complex.

3. Arguing from the substance to the genes

The alternative mode of approach to the problem of the production of substances by genes is to identify the substances and to try to trace them back to the genes. A very great deal of information has been obtained in this manner, but there is a fundamental difficulty in deriving a complete theory in this way, because we can never be certain, as we trace a substance back through its precursors, that we have reached the last stage from THE SYNTHESIS OF NEW SUBSTANCES 377

which we are justified in leaping direct to the gene itself: there is always the possibility, which is indeed often a probability, that there are several intervening steps between the gene and the most deep-lying precursor which we have been able to find. . Most gene-controlled substances which can be easily identified are found in the cytoplasm, and are probably produced in it, so that the genes must be involved only at second hand in their formation. Direct evidence of the production of developmentally active substances by the nucleus itself, or its immediate neighbourhood, is, however, available in some cases. One of the most striking of these occurs in the unicellular alga Acetabularia (Fig. 17.3). During most of its life-cycle this organism consists of a rhizoid, which is attached to the ground, from which arises a stalk which terminates in an umbrella-shaped hat. There is only a single nucleus, although the whole alga may attain the size of several centimeters or more. Haemmerling (1934, 1953) showed that if the hat is removed, a new one will regenerate. He then cut off the nucleus-containing rhizome from an alga of one species (A. mediterranea) and substituted a similar piece con


Nuclear grafts in Acetabularia. (A) Acetabularia mediterranea; the stem has been somewhat shortened in the drawing; the single nucleus lies in the thizoid at the bottom. (B) A. Wettsteinii. (C) the Wettsteiniilike ‘hat’ regenerated from a short piece of mediterranea stem grafted on to the nucleuscontaining rhizoid of Wettsteinii. (After Haemmerling 1934.) 378 PRINCIPLES OF EMBRYOLOGY

taining the nucleus of a different species (A. Wettsteinii). When the hat was now removed, the important point emerged that the new one which regenerated had the characters of Wettsteinii, that is to say of the nucleus lying at the base of the alga and not of the stalk to which the regenerate was attached. It is clear that the nucleus (presumably the genes contained in it) has caused the production of some substance which controls the morphogenesis of the regenerate. Unfortunately nothing is known of the chemical nature of the substances in question.

It has recently been found, e.g. by Mirsky (1951), that different tissues characterised by their richness in particular substances may contain these substances not only in the cytoplasm but also in the nuclei. For instance, haemoglobin is found in the nucleus in the early stages of the development of red blood corpuscles. We have already mentioned such facts as examples of the differential activation of genes by their associated cytoplasm; the point which is being made here is that it also suggests that the substances concerned are manufactured in close proximity to the genes themselves and may be immediate rather than secondary gene products.

4. Genes and enzymes

The substances which have been identified in nuclei in this way are mostly enzymes. There is a good deal of other evidence which indicates that genes may frequently operate by means of their effect on cellular enzymes, although as we shall see, these are more probably formed in the cytoplasm rather than directly by the genes. One of the earliest cases in which the effect of a gene could be described in biochemical terms was that of alkaptanuria in man; the homozygote for a certain recessive gene is unable to oxidise homogentisic acid to allantoin, and the former is therefore excreted unchanged in the urine (Garrod 1923). It seems that the normal allele of the alkaptanuric gene is essential for the production of the enzyme which brings about the oxidation. Several other similar cases have been described in mammals, and in recent years very many have been discovered in lower organisms such as moulds, fungi, yeasts, bacteria, etc. (Reviews: Catcheside 1951, Beadle 1949, Horowitz 1950, Haldane 1954). Normal strains of these organisms can grow on relatively simple media, from which they can synthesise all the substances necessary for their continued existence. If a strain, say of the fungus Neurospora, is x-rayed or otherwise caused to mutate, and the haploid spores produced are tested for their nutritional requirements, many mutants will be found which cannot grow unless certain specific substances are added to the basic medium, It is concluded that some step in the chain of reactions, which in THE SYNTHESIS OF NEW SUBSTANCES 379

the normal strain would lead to the synthesis of the substance concerned, depends on a gene which has mutated and is no longer able to carry out its proper function.

By collecting and testing strains with different nutritional requirements, much may be learnt about the chains of reactions. An example of this is illustrated in Fig. 17.4. The chain of reactions by which arginine is synthesised may be broken by mutations 1 to 7. Mutants 1 to 4 will grow if supplied with any of the substances which occur later (ornithine,

ornithine citrulline arginine NH CONH, NH, S

NH, 1,2,3,4 (CH); 586 5 NH 7 , CoNH —» proteins

CHNH, (CHa) NH |

COOH a (CH,); |


COOH co wee 2 , urease + C=O urea 2NH; NH, FIGURE 17.4

Metabolic pathways leading to the synthesis of arginine, with indications of the points where different genes in Neurospora (1, 2, 3, 4, 5, 6, 7) interfere.

citrulline or arginine); 5 and 6 can grow if provided with citrulline or arginine, but ornithine does not make good their deficiency, while 7 requires arginine and cannot make do with either of the other two substances. Very many similar situations have been discovered.

Since many of the steps in these sequences are known or believed to be carried out by means of enzymes, it is natural to assume that the fundamental activity of the genes involved is the production of these enzymes, and that when the gene mutates the enzyme is either not produced at all or appears in an inactive form. In most cases this suggestion remains a hypothesis; it is rarely that the enzymes have actually been investigated, and when this has been done it has sometimes been found that the relevant one is not absent from a strain in which some reaction-sequence is interrupted, but is present though not effective (Wagner 1949). Nevertheless the postulated gene-enzyme relationship has been generalised and made 380 PRINCIPLES OF EMBRYOLOGY

into the so-called ‘one-gene-one-enzyme’ theory. This supposes that all genes operate through the medium of enzymes, and that each gene is connected in the first place with one and only one enzyme. In one form of the theory the gene is supposed actually to produce the whole enzyme; in another the suggestion is only that the gene determines the specificity of the enzyme, and thus its ability to react, and its efficiency in reacting, with the appropriate substrates.

This hypothesis has been widely believed in recent years, and even when some doubts are expressed as to its adequacy, it has usually been given credit for having stimulated a great deal of valuable work. Actually, however, it is probable that the stimulus to the work came rather from the experimental technique of identifying mutations which lead to the blockage of a series of synthetic reactions rather than from the one-gene-oneenzyme hypothesis itself. In fact, almost the first problem which the hypothesis would raise is whether a given gene necessarily controls the formation of the same enzyme in different tissues, in which it is reacting with different cytoplasms. This question, however, still remains quite unanswered and hardly tackled, not, surely, because it is far removed from theory but because it demands a different experimental approach.

The gravest criticism of the one-gene-one-enzyme theory is that it draws its support almost entirely from studies of unicellular or very primitive organisms and thus leaves out of account of the whole range of phenomena involved in regionalisation, which may or may not fall into line with it. Even within the realm of the micro-organisms, it seems that at the present time the theory is beginning to appear as an oversimplification (cf. Haldane 1954). However, work of the kind associated with it has led to the production of numerous genes which have thrown light on a wide variety of problems. For instance, it has been shown that a gene which has mutated to an apparently inactive form may many generations later mutate back again, and the corresponding activity reappears in the cells. This shows that a gene may continue to multiply in a sequence of dividing cells even though it shows no signs of activity; though of course it remains doubtful whether the gene is truly inactive or is continuing all the time to produce an enzyme of altered specificity.

Again, this material provides some relatively clear-cut examples of secondary interactions between gene-controlled processes of the kind which seem necessary as a basis for a general theory of development (cf. Chapter XIX). Thus in Neurospora a certain gene blocks the formation of isoleucine and this leads to the accumulation of its precursor, which cannot be utilised but increases in concentration until it inhibits a different but connected reaction by which a-ketoisovaleric acid is changed into THE SYNTHESIS OF NEW SUBSTANCES 381

valine (Bonner 1946). Another type of interaction is exhibited by the fact that in certain strains of Neurospora and Aspergillus which require an external source of L-arginine, an addition of L-lysine to the medium reduces the effect of the added arginine; and the same competitive inhibition between the two substances is shown by a lysine-requiring strain (cf. Pontecorvo 1950, Emerson 1950).

Finally, we may quote from another field a biochemical example of competition between gene-produced substances (or, less probably, the genes themselves) for a common substrate. One of the earliest attempts to link up gene action with known chemical compounds was the investigation of the genetic control of flower colour (Lawrence and Price 1940, Haldane 1941, 1954). Genes were identified which caused various precisely known chemical changes in the constitution of the coloured substances. Probably these genes act by controlling the formation of enzymes; the evidence is no better, and not much worse, than it is for the similar hypothesis in the Neurospora work. Now in crosses involving a number of genes affecting the various different classes of pigments (anthocyanins, flavones and chalkones) it was found that there were interactions in which all the different types of substance were involved. When much anthocyanin was formed, this led to a reduction in the amounts of flavone and/or chalkone. Thus the different gene-activities competed, with efficiencies corresponding to the number and strength of the genes involved, for a limited amount of a common substrate from which the compounds of all those types were derived (Lawrence 1950).

5. The synthesis of proteins

Since most, if not all, enzymes are proteins, the evidence from genetics that genes control the formation of enzymes should be linked with such general information as we have about the synthesis of proteins in cells. Although the nature of the chemical processes involved in this synthesis is still almost entirely obscure, there is quite a large amount of rather indirect evidence on the role which various parts of the cell structure play in the production of proteins. Most of this does not come from investigations which have used embryos as the experimental material (for which see ten Cate 1953 and Gustafson 1952) but from studies in the general field of biochemistry and physiology.

It is, in the first place, fairly generally agreed that the nucleic acids participate in an important manner in protein synthesis and indeed are probably essential for it. The synthesis of new chromosomal proteins, which is involved in the reduplication of the genes before cell division 382 PRINCIPLES OF EMBRYOLOGY

takes place, never occurs in the absence of desoxyribose nucleic acid. In the synthesis of cytoplasmic proteins, it is ribose nucleic acid which is thought to play the active part. Chemical analyses always demonstrate an unusually high concentration of RNA in rapidly growing cells or in those which are actively secreting protein (e.g. some glands, hair-forming cells, etc.). This RNA is largely located in the cytoplasm, but there is often also a considerable enlargement of the nucleoli, which are rich in RNA. For example, nucleoli are absent in the cleavage cells of the amphibian neurula, but appear at about the time of gastrulation, when there is evidence that specific proteins begin to be produced.

There are at present two main theories about the processes of protein synthesis in embryonic or developing cells. The first is that of Caspersson (Reviews: 1947, 1950). It is based chiefly on studies which use spectroscopic methods to take advantage of the fact that the purine and pyramidine bases incorporated in the nucleic acid molecule have very characteristic absorptions in the ultra-violet; this makes it possible for the nucleic acids to be identified within living cells, although it must be pointed out that there are considerable technical difficulties and the method has come in for a good deal of criticism. From his observations, however, Caspersson has come to the following conclusions. The euchromatin, which consists largely of histone and DNA, synthesises replicas of itself and also produces other complex proteins, which could be the agents through which geneaction is exerted: however, the spectroscopic evidence does not suffice to suggest much about them. The heterochromatin (or, more generally, the ‘nucleolus-associated chromatin’) which contains an important proportion of RNA, is supposed to control the nucleic acid metabolism of the whole cell. It also produces proteins, which tend to be rich in diamino-acids. These accumulate in the nucleolus, and diffuse from there to the nuclear membrane. On the outside of this, an intensive production of RNA-protein takes place and this is the main source of the cytoplasmic proteins, which are thus supposed to be formed in the immediate neighbourhood of the nucleus (Fig. 17.5).

The other main theory is that of Brachet (cf. 1950, 1952). It differs from that of Caspersson in being derived from a large variety of biochemical, cytological and embryological investigations rather than from a single technical method such as spectroscopy. In its conclusions it lays much more stress on the role of the various types of cytoplasmic particle in protein synthesis. Brachet supposes indeed that the ribose nucleotides formed by the nucleolus (or heterochromatin) do not merely combine with proteins in the neighbourhood of the nuclear membrane, but take part in the formation of microsomes scattered throughout the whole THE SYNTHESIS OF NEW SUBSTANCES 383

cytoplasm, and that it is at these particles that the main synthesis of cytoplasmic proteins occurs.

There is as yet, perhaps, no evidence which finally settles the question of whether cytoplasmic synthesis is directly under the control of the nucleus or whether the microsomes are essentially involved. The importance of the nucleus, either at first or second hand, can of course not be


Protein synthesis according to Caspersson. A, the ‘nucleolus-associated

chromatin’, containing ribo-desoxy-nucleotides (RDN), proteins (P), di amino-rich proteins (DP) and perhaps ribose-nucleotides (RN). B, the

nucleolus, containing DP and smaller amounts of RN. C, the nucleus, in

which there is a gradient of P and DP towards the nuclear membrane D.

In the cytoplasm E there is a gradient of P and RN from the nuclear membrane outwards. (After Caspersson 1950.)

denied. For example, Weiss and Hiscoe (1945) have shown that in a neuron growth does not take place throughout the enormously elongated axon, but synthesis occurs only in the cell body in the neighbourhood of the nucleus; the new cytoplasm flows from this region towards the tip of the fibre, and if the axon is constricted, the flow becomes dammed up and a swelling appears proximal to the constriction (Fig. 17.6). The cell body is, however, also the region in which the cytoplasm is most basophilic and probably the site of the main concentration of microsomes, so 384 PRINCIPLES OF EMBRYOLOGY

that it remains quite possible that the influence of the nucleus is indirect and mediated through them. Again, Brachet and Chantrenne have shown by radioactive tracer studies that if one removes the nucleus from the large unicellular alga Acetabularia (cf. p. 377), protein synthesis eventually decreases in the non-nucleated fragment (cf. Brachet 1954). But it is remarkable that the activity continues unaltered for almost a fortnight and remains very considerable for much longer; from this they draw the


Diagram of nerve regeneration. Rows A to E show the normal process when the nerve is simply cut; regeneration takes place from the proximal portion. Rows F, G, H show that if, after the stage of row D, the fibre is constricted, the new cytoplasm being synthesised in the proximal (nuclear) end of the cell forms a swelling. When the constriction is removed (J) this cytoplasm gradually spreads distally. (After Weiss and Hiscoe 1948.)

conclusion that the nuclear control of protein synthesis is indirect. The final proof that the microsomes take part in the process, and are the actual site of the new production of protein, would be given if they could be transplanted to some unusual location, say to another cell of different developmental fate, and it was found that the protein characteristic of the transplanted material was produced in the new position. Attempts to do this with isolated microsomes have so far been unsuccessful. The results of high-speed centrifugation of ascidian eggs (p. 114) or of echinoderm eggs (p. 90) may, however, probably be interpreted in this way, since THE SYNTHESIS OF NEW SUBSTANCES 385

the particles moved under these forces would seem to be comparable with those usually considered as microsomes. The role played by microsomes in the synthetic activity of echinoderm embryos, and their relations with the mitochondria, have been discussed earlier (p. 90).

It will be seen that neither in Caspersson’s nor in Brachet’s theories is it supposed that the synthesis of proteins occurs actually at the gene inside the nucleus. If enzymes are sometimes produced in this way (p. 355), which is by no means certain, that can hardly be the general rule of protein synthesis. One suggestion has been made (e.g. Wright 1945, Waddington 19394), however, which minimises the difference between synthesis at the gene and synthesis in the cytoplasm. According to this, the genes directly produce substances which are replicas of themselves in most respects (or possibly in all except for the connecting links which hold the genes together in the chromosome); and it is supposed that these replicas pass into the cytoplasm and there control the synthetic processes. This hypothesis has the merit of simplicity, but the evidence for it is slight; the fact that synthetic activity disappears or diminishes when the nucleus is removed suggests that the postulated gene-replicas cannot at all fully take the place of true nuclear genes and it is therefore likely that they cannot actually be replicas in the full sense of the word. It may very well be the case, however, that the immediate products of gene activity have a considerable chemical resemblance to the genes or to some part of them. Possibly the genes produce, as a first step, a product which resembles the protein which, in the chromosome, is combined with DNA, and this, passing into the cytoplasm, becomes associated with RNA, either near the nuclear membrane as Caspersson suggests or in the microsomes of Brachet.

It will be seen from the discussion in this chapter that in spite of the very large effort which has been devoted recently to the attempt to discover how genes operate, and the important body of interesting results which have been gathered, there has really been rather little progress towards the solution of the problem which has been put in the centre of the stage. This may be due to the inherent difficulties of the field; but the biologist who is not primarily a biochemist may be tempted to wonder whether the problem is not perhaps being envisaged in too simple terms. We have tended to think both of genes and of their products as definite and discrete particles, of, perhaps, the dimensions of a protein molecule or a little more. Possibly the active participants in gene-operations are actually of a higher order of complexity than this. As Goldschmidt has urged, and as we have seen above, there is considerable evidence which could be taken to support the idea that the active units in chromosomes 386 PRINCIPLES OF EMBRYOLOGY

are relatively long structures, rather than ‘point-genes’; and electron microscope studies on cytoplasmic structure (Fig. 17.2) suggest that we may have tended also to under-estimate the size and complexity of the active agents in protoplasm. We may, perhaps, find that it is necessary to think of gene action in terms not of enzymes or other protein molecules as we know them in solution, but of extended protein sheets and fibres, which may have properties which largely escape our present biochemical methods. Possibly it is in this way that we shall find an explanation both for the very large number of genes which affect the development of an organ and for the extreme precision with which they control it.


Brachet 1952a, b, Haldane 1954, Chapter 2, Goldschmidt 1951, Muller 1947, Pontecorvo 19524, b, Spiegelman 1948, Schultz 1952, Wright 1945.