Cybernetic interactions in epigenetics

3 CYBERNETIC INTERACTIONS IN EPIGENETICS GAJANAN V. SHERBET Chester Beatty Research Institute, Institute of Cancer Research, Royal Cancer Hospital, London

CONTENTS INTRODUCTION

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NUCLEOCYTOPI.ASMICINTERACTIONS (a) Cytoplasmic heterogeneity: the ooplasms (b) Mutual effects of the cytoplasm and the nucleus (c) The biochemical nature of heterogeneity (d) Structural organization and differentiation

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EMBRYONICINDUCTIONS (a) The inductive stimulus as effector molecules in gene derepression (b) A possible role for hormones (c) Synthesis of specific RNAs as a controllingfactor

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IV.

ROLEOF COMPETENCEIN DIFFERENTIATION The nature of competence

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V. DIFFERENTIATIONIN GRADmNT SYSTEMS

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ACKNOWLEDGEMENTS

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3 CYBERNETIC INTERACTIONS IN EPIGENETICS GAJANAN V. SHERBET Chester Beatty Research Institute, Institute of Cancer Research, Royal Cancer Hospital, London

I. INTRODUCTION Epigenetics is the branch of biological science which deals with the causal analysis of development. Cellular differentiation is an integral part of developmerit and epigenetics has been concerned with resolving the question of the causal mechanism of cellular differentiation. In the early stages of development the zygote divides repeatedly and becomes multiceUular. Potentially all the cetls should possess equivalent genetic equipment. Yet different parts of the multicellular embryo give rise to different cell types which are characterized by recognizably distinct proteins. This is obviously a result of the differential activity of genes. It has become apparent in recent years that the factors of fundamental importance ate the cybernetic interactions between the component parts of a developing embryo. These interactions can be considered under two major subdivisions, viz. nucleocytoplasmic interactions and embryonic inductions. Tins paper is devoted to a discussion of these interactions.

II. NUCLEOCYTOPLASMIC INTERACTIONS It was suggested by Morgan as early as in 1934 that a differential activity of genes caused by the heterogeneity of the cytoplasm itself brings about a progressive change in the character of the cytoplasm. This argument seems to be sound even today, for example, as modified and developed by Waddington (1956, 1962). Alternative hypotheses such as deletion of part of the genetic material, etc. have also been suggested (see Markert, 1964). (a) Cytoplasmic Heterogeneity: the Ooplasms The former hypothesis is supported by a large amount of experimental evidence, which can now be discussed. The basis on which the hypothesis rests is the fact that in a number of invertebrates and vertebrates the cytoplasm of the egg cell has been shown to be heterogeneous. The molluscan eggs, for example, are characterized by the presence of different ooplasms which differ in their inclusions and staining properties from the rest of the cytoplasm (see Raven, 91

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1958, 1963). In Dentalium, Wilson (cited by Waddington, 1956) has demonstrated the occurrence of the polar lobe material. This material is rather distinctive. Before undergoing cleavage this separates into a polar lobe. The egg then divides. Subsequently the polar lobe fuses with one of the blastomeres. In ascidians similar differences in the cytoplasm have been shown to exist (Reverberi, 1961). The grey crescent of the amphibian egg is another example of the heterogeneity observed in the cytoplasm. The cells of the embryo which contain this grey crescent material constitute the organizer region which in subsequent development induces the formation of the neural axis. Distinct formation of ooplasms in the oocyte of Ambystoma has been recently demonstrated (Harris, 1964). Qualitatively distinct ooplasms thus occur in the zygote. What such qualitative heterogeneity might mean in chemical terms will be discussed later. (b) Mutual Effects of the Cytoplasm and the Nucleus Nuclear transplantation experiments have shown that during development the nuclei gradually lose their potentialities. Development is accompanied by nuclear differentiation (Briggs and King, 1955; Moore, 1960, 1962; Gurdon, 1962, 1963; Fischberg and Blackler, 1963; Simnett, 1964), the nucleus becoming more and more specialized in its functions (Brachet, 1957). Fischberg and Blackler have shown that these nuclear changes are both stable and heritable. In the opinion of Stern et aL (1952) the changes involved are chemical as well as morphological. Gurdon (1963) believes that these nuclear changes are stable genetic changes. Their onset is reported to be earlier in the ectoderm than in the endoderm (Simnett, 1964) and seems to correspond to the onset of stability of the processes of determination in these germ layers (see Saxen and Toivonen, 1962). Visible changes in the chromosomes brought about by cytoplasmic factors have been very convincingly demonstrated in the salivary gland cells of Drosophila and Chironomus. Beerman (1961) showed that appearance of a Balbiani ring can be correlated with the presence of certain specialized granules in the cytoplasm of the gland cells. Kroegar(1960)transplantedsalivaryglandnucleiofDrosophila into cytoplasm of eggs and found puff formations which were not noticed in normal development. If changes are brought about in the cytoplasmic environment experimentally, new puffing patterns could be induced (Clever, 1961, 1963; Ritossa, 1962; Berendes et aL, 1965). It will be of interest to note that ooplasms sometimes become active only after they interact with nuclei. Thus, Seidel (1929) showed that the posterior region of an insect egg which constitutes the formation centre becomes active only after the nuclei reach it and, seemingly, after such interaction produces a substance which diffuses into the anterior regions and causes the differentiation of the embryo. The involvement of nucleocytoplasmie interactions during the differentiation of oral structures has been shown in the case of some protozoa also (Tartar, 1961 ; de Terra, 1964).

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(c) The Biochemical Nature of Cytoplasmic Heterogeneity What is the nature of the initial heterogeneity of the cytoplasm and the later diversity resulting from these interactions ? The answers to these questions are still in the realm of speculation. The initial heterogeneity could be visualized in chemical terms. The existence of respiratory gradients, RNA gradients, specific distribution of-SH groups, glycogen, etc. have been demonstrated years ago (see Saxen and Toivonen, 1962, for a recent review). Qualitative and quantitative differences in enzyme contents have been shown to exist (Ten Cate, 1953). The presence of many hormones in the eggs has also been demonstrated. Some type of localization of gonadotropic hormones seems plausible (Sherbet, 1963). Deuchar (1963) has discussed a considerable amount of data on the distribution of free amino-acids. There seems to be no doubt that variation in the free amino-acid pool and distribution of some of them in the developing embryos are quite characteristic and meaningful from the point of view of differentiation. Apart from these factors there is probably a significant distribution of the raw materials required for biosynthetic processes. It is not known what factors present in the ooplasms cause differential gene activation. But we do have some idea as to what results from such differential activation. It seems to be mainly a synthesis of the immediate gene products, viz. the RNAs, having, as is now well established, distinct functions in the biosynthesis of proteins. It is significant, therefore, that the puffs in the polytene chromosomes which seem to be induced by the environmental cytoplasm are sites of intense RNA synthesis (Swift, 1962; Ritossa, 1964; Ritossa et aL, 1965; Pelling, 1964; Berendes et aL, 1965). The formation of these RNAs is inhibited by actinomycin treatment which causes a regression of the puffs (Laufer et aL, 1964). Similarly, ecdysone-induced puffs could be inhibited by actinomycin. The RNAs, therefore, are presumably of the messenger type (Clever, 1964). Once a differential gene activity is initiated, as shown by Waddington (1957), if the processes of biosynthesis are autocatalytic small initial differences can bring about divergent and important differences between the different regions of the cytoplasm. For example, suppose two substances A and B give rise to P, and B and a third substance C give rise to Q. If these synthetic processes are autocatalytic, i.e. the very presence of end products P and Q catalyse the reactions, a slight difference in the quantity of on~ of the reactants would bring about proportionately very large quantitative differences in the end products. If amount of substance A is slightly greater, product P will be formed in a proportionately greater amount (for a mathematical derivation see Waddington, 1957). A distribution of the raw materials might thus direct the synthetic processes. By such a process specific protein (enzymic?) patterns might be built up in different regions of the embryo which ultimately bring about in a progressive manner synthesis of different tissue-specific proteins. But this has to await the arrival of different type of stimulus. Possibly, each ooplasm is concerned with

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the activation of a set of related genes. It seems thus that the regional differentiation of the cytoplasm which results from cybernetic interactions between it and the nucleus is essentially a chemodifferentiation (see Fig. 1). Instances of enzyme adaptation in embryos have been recorded in the formation of adenosine deaminase {Gordon, 1952) and succinic dehydrogenase (Boell, 1949). Enzyme adaptation may be caused by a distribution of simple substrate raw materials and may provide some explanation for the enzymic heterogeneity obtaining in developing embryos.

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FIG. I. A diagrammatic representation of Interactions between the nucleus and the cytoplasm. I is the interaction between the heterogeneous cytoplasm and the nucleus whlch probably brings about a differential activity of the genes. This might imtiate interaction II between genes. The activity of the genes culminates in the synthesis of primary proteins (III). A feedback mechanism may exist between the cytoplasm and the nucleus at this stage (IV). The primary proteins are responsible for the synthesis of specific phenotypic proteins which may react on the primary proteins (V). The numerals do not imply any sequence of interactions.

Different enzymic complements seem to be associated with different cell types at any one time in the course of development. Association between differentiating neural retina and enzymes such as alkaline phosphatase, carbonic anhydrase, cholinesterase, glutamotransferase, etc. has been studied in some detail (Moog, 1958a). The appearance and increase in alkaline phosphatase in the duodenal epithelium is apparently controlled by the secretion of adrenal cortical hormones. It has been suggested that the hormones do not directly accelerate its synthesis but hasten the differentiation of the epithelium and that the synthesis of the enzyme is dependent upon the level of its differentiation (Moog, 1958b). Differentiation of the nervous system is accompanied by an enormous inciease of cholinesterase (Boell and Shen, 1950; Boell et aL, 1955). In sea-urchin embryos two series of enzymes appear in different phases of development. Alk~ine phosphatase, dehydrogenases, glutaminase I, cathepsin

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II, apyrase and cholinesterase show a uniform level during the cleavage phase and increase at the late blastula and gastrula stages. A second series of enzymes comprising aldolase, adenosine deaminase, phonylsulphatase, acid phosphatase, dooxyribonuclease, etc. showed no increase up to the pluteus stage. It is clear that some enzymes show an increase at the time of onset of visible differentiation (see Gustafson, 1954). Investigations have revealed a significant distribution and variation of cytochromes, succinic dehydrogenase, cholinesterase, alkaline phosphatase, phenolases, etc. in the ascidians and have been summarized by Reverberi (1961). All these enzymes with the exception of cholinesterase show a positive pattern. The phenolases particularly offer an interesting correlation. There are two pigmented organs in the brain region, viz. the eye and the otohth. The melanin pigmentation is formed from non-pigmented precursors which are "activated" by some enzyme. This enzyme seems to make its appearance during neurulation when the formation of the brain is also induced. Minganti (1951) showed that if the animal tier cells are separated from the vegetal tier at the 8-celled stage, the animal tier gives rise to a larva which not only lacks a brain but the melanin precursors and the oxidative enzyme also. Reverberi (1961) has suggested, therefore, that the enzyme formation is "conditioned" by the brain. Wallace (1961) has reviewed some data dealing with the localization of enzymes or the increase in their activity in specific regions, in other words, the enzymic patterns as correlated to the state of differentiation in amphibian embryos. His own experiments indicate that certain enzymes show increased activity around late gastrula or neurula stages while others increase only at the hatching stage. A correlation between pattern and differentiation seems to exist for isozymes too. Each tissue seems to possess its own isozymic pattern which changes during embryonic development until the adult pattern is attained. The existing isozymic pattern can be taken to reflect the state of differentiation (Markert and Moller, 1959). (d) Structural Organization and Differentiation It is pointed out by Ten Cate that owing to structural conditions of a cell certain enzymes might be present in inactive state in the early embryo. The enzymes might need surfaces or structures to become attached which will give them the necessary localization, concentration, etc. This will also facilitate co-operative action among the various enzymes of a system and in turn facilitate protein biosynthesis. It is to be expected therefore that a correlation between the structural organization of a cell and its state of differentiation should exist and has actually been brought to light by the electron microscope. The current view of protein synthesis holds that the genetic information is carried to the cytoplasm by the agency of specialized RNAs which attach themselves to the ribosomes, the latter being the actual sites of protein synthesis. Waddington (1963a) believes that the endoplasmic reticulum may play an important role in linking together different synthetic processes. For a single type of cell not only synthesizes a protein which is characteristic of it but many

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related proteins as well. All these syntheses have to be integrated to achieve diffexentiation. It is understandable therefore that a cell which is differentiating also builds up the necessary structural organization. According to Palade (1958) a cell at the beginning of differentiation contains very little of the reticulum but numerous cytoplasmic particles. In the course of differentiation there occurs an elaboration of the reticulum and the particles become associated with it. Such organization of the reticulum has been described in differentiating pancreatic cells. In early embryos also very little of organized endoplasmic reticulum seems to be present. Cells which are differentiating have characteristic types of the reticuium. If in the subsequent life-history of a cell a change in the type of its product is involved it is also accompanied by a change in the organization of the reticulum IWaddington, 1963b). It seems also probable that genic mutation is reflected in the structural organization. Waddington (1960) has pointed out the abnormal organization of cytoplasmic structure in some component cells of the ommatidium of a mutant form, the roughoid g, of Drosophila. A further example is provided by the presumptive neural tissue cells which possess a loose endoplasmic reticulum. The latter becomes more complex when they begin to differentiate into neural cells in response to the inductive stimulus received from the chorda mesoderm (Saxen and Toivonen, 1962).

III. EMBRYONIC INDUCTIONS The processes of embryonic development can be analysed into a series of inductive phenomena which appear in a temporal sequence. Every process of induction probably has an acting system and a reacting system. The reacting system is ultimately moulded into a particular cell type. The acting system, which now seems to be comparatively less important and rather unspecific, gives some sort of a stimulus, probably a chemical one, which sets off a chain of reactions that culminate in the synthesis of specific proteins or metabolic enzymes characterizing a specific cell type. A large amount of work has been done to study the nature of the inductive stimulus. Since it is the reacting system which becomes physically moulded the obvious effect of an inductive stimulus seems to be activation of the genetic material of the reacting system. (a) The Inductive Stimulus as Effector Molecules in Gene Derepression Jacob and Monod (1961, 1963) postulated that the regulator genes produce allosteric protein molecules which act as repressors and which can reversibly combine with effector molecules. When the repressor is bound in this way the structural genes can synthesize RNAs (Fig. 2). The DNA could be supposed to be repressed on account of binding to some inhibitor macromolecules. When the repressor molecule combines by its allosteric site with the effector the reactive site changes such that it can now combine with macromolecules which are inhibiting the synthesis of RNAs.

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CYBERNETIC I~rERAC'nONS IN EPIGENETICS

RG

0

SG

SG

V M essenqTerRNAs

11

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Regula,ory metabolite (effector)

Enzym,c proteins

FIG. 2. Schematic representation of the Jacob and Monod theory of enzyme induction. R G : Regulator gene; O: operator gen¢; SG: structural gen¢. The repressor is postulated to b¢ an allosteric molecule (see Fig. 3).

Earlier studies have established the fact that during embryonic induction a transfer of material occurs from the inductor into the reacting system (Ficq, 1954; Waddington and Sirlin, 1955; Sirlin and Brahma, 1956, 1959; Waddington and Mulherkar, 1957; Pantelouris and Mulherkar, 1957; Grobstein, 1956, 1959, 1961; Koch and Grobstein, 1963). The process of induction could be brought into line with the Jacob and Monod theory if one postulates that the molecules transferred into the reacting system act as effectors in the above described circuit and combine with the allosteric repressor molecules and cause a derepression of the concerned structural genes and initiation of synthesis of specific messenger RNAs (see Fig. 3). Waddington (1962) first suggested that the inductive stimulus either impinges directly on the structural genes (genotropic action) or on the regulator gene product (plasmotropic action). Some unpublished results obtained by the present author in experiments on the effects of calf thymus histones on early chick development seem to suggest that the inductive stimulus acts in the

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(a) (b) FIG. 3. ,4 possible scheme of events in an embryonic induction. A: A competeat cell of the reacting system. B: Interaction between the reacting cell and the inductor resulting in the process of induction. RP: Regulator gene product (repressor allosteric molecule); GI: (3ene inhibitor; F_S: Effector inductive stimulus and MRP: modified regulator gene product (Effector-repressor complex). Other notations as in Fig. 2.

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plasmotropic fashion. This suggestion is compatible with certain observations on the process of induction made in Waddington's laboratoly such as the locahzation of steroids and hydrocarbons in the cytoplasm; large-scale transfer of labelled material from heterogeneous inductors to the cytoplasm of the reacting cells. However, with natural inductors transfer of material seemed to be mainly directed towards the nucleus (Waddington, 1962, pp. 26-8). Since transfer of material has not been shown to take place exclusively into the nucleus, the presence of labelled material in the cytoplasm cannot be simply viewed as accidental or transitory and without significance. These experiments no doubt show that the question of whether the inductor substance acts genotropically or plasmotropically is still open. (b) A Possible Role for Hormones Since specific RNAs are held to be immediate gene products a control or experimental alteration of genic activity should be possible by controlling or altering the synthesis of RNAs. Hormones have been investigated intensively from this angle. Kidson and Kirby (1964) observed selective alterations in rapidly labelled RNA profiles on injecting many hormones. Korner (1962, 1964) demonstrated that growth hormone acts by regulating the rate of messenger RNA synthesis. Talwar and Segal (1963) have shown that estradiol and chorionic gonadotropin (HCG) cause maturation of gonadal tissues through an initial stimulation of messenger RNAs. The same mechanism has been shown to operate in case of the thyroid hormone (Tata, 19635. Callantine et aL (1965) found that follicle-stimulating hormone (FSH) and HCG cause a significant increase in ovarian RNA. The increase is also accompanied by an increase in the ovarian weight. FSH has been found to stimulate accumulation of utilizable and non-utilizable amino-acids (Ahren and Rubinstein, 1964). Such accumulation continues even in the presence of puromycin which inhibits protein synthesis. Studies were also made by the author on the capacity of FSH to induce differentiation. These studies were stimulated by the work of Mangold et aL (1965) who found that hypophyseal extract pellets produced neural inductions. This was followed up by grafting pieces of fresh anterior pituitary glands from a mammalian source into early chick embryos and very good neural inductions were obtained (Sherbet, 1962). Pieces of amphibian anterior pituitary also produced neural inductions. This capacity of induction was analysed in a series of experiments. Different solvents were used in order to achieve a kind of fractionation. For example, 0.5 saturated ammonium sulphate (0.5 SAS) precipitates all hormones of the anterior lobe and the gland retained its capacity of induction after treatment with 0.5 SAS. 50 per cent pyridine removes FSH, LH and TSH and the gland lost its capacity following pyridine treatment. LH was eliminated on the basis of 2-5 per cent TCA treatment. Between FSH and TSH the former seemed the more likely candidate because ovary is the target organ of this hormone. Apart from that a qualitative and quantitative estimation of the inductions

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indicated that the female gland is more potent than the male in one season. The gland from a breeding female was more potent than that from a non-breeding female (Sherbet, 1963). 0.5 SAS and 50 per cent pyridine affected the inductive capacity of the Hensen's node in similar manner (Lakshmi and Sherbet, 1962). Recently it was found in direct experiments that if FSH is incorporated in agar medium and posterior pieces of early chick blastoderms were cultivated on it, they differentiated into neural tissue, notochord-like tissue and aggregations of mesoderm resembling somites. Control explants similarly cultivated on agar without FSH differentiated only into large masses constituting bands of mesodeim (Sherbet and Mulherkar, 1963). In another set of experiments early chick embryos were treated with FSH in vitro and their inductive capacity was tested. It was found that FSH confers some capacity on postnodal primitive-streak pieces to cause neural inductions and also supports their differentiation into mesenchyme and somites (Sherbet and Mulherkar, 1965). Preliminary experiments now indicate that HCG causes a preferential incorporation (or transport 7) of labelled glycine into neural tissue and extra-embryonic ectoderm (Sherbet and Lakshmi, unpublished). More intensive investigation in this direction by autoradiographic and immunological methods is required. With this background we cannot resist the temptation to speculate that FSH might become localized in the developing ovum and at a later stage in development act as an inductive stimulus in primary embryonic induction or any subsequent inductions. It is relevant to state in this connexion that some follicular hormones have been detected m early chick embryos (Mitskevich, 1957). (c) Synthesis of Specific R N A s as a Controlling Factor If hormones do regulate the rate of messenger synthesis and the inductive stimuli of embryonic inductions act in the same manner, it could be argued that regulation of such synthesis might be one of the controlling factors in development. If we interfere with such synthesis we should be able to obtain specific developmental abnormalities. A number of investigations using actinomycin have been reported. Flickinger (1963) found that actinomycin D causes malformation of different organs of the embryo depending upon the stage of development at which treatment is commenced. Brachet and Denis (1963) and Brachet et aL (I 964) found that the antibiotic causes developmental aIrest at gastrulation, microcephaly, improper differentiation and degeneration of the nervous system in certain amphibian embryos. The present author's unpublished experiments have indicated that actinomycin D causes arrest of development of chick ~mbryos at stage 4--5 (Hamburger and Hamilton, 1951) or abnormal brain formation. It has been proved that this antibiotic interferes specifically with the synthesis of DNA-dependent RNAs at low concentrations (Reich et aL, 1961, 1962; Goldberg and Reich, 1964). Curiously enough, histone fractions obtained from calf thymus have been found to produce similar effects in chick embryos (Sherbet, unpublished). It is interesting to note that histones have also been shown to inhibit DNA-dependent RNA synthesis (Huang and Bonnet, 1962; Barr and

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Butler, 1963; Allfrey et al., 1963). Though the effects produced by actinomycin and histones are similar it is probable that the mechanisms of their action are different. However, it should be emphasized that in both cases the effects probably are brought about by inhibition of DNA-dependent RNA synthesis. The arrest of development at gastrulation or plimitive-streak stage in amphibia or chick and also the all-or-none response shown by gastrulating embryos of Planorbis exustus (Sherbet and Lakshmi, 1964a, b) seem to indicate that synthesis of important messenger RNAs begins at gastrulation. The stage of gastrulation is a crucial one in development and involves high synthetic activJty which accompanies the rapid processes of differentiation. It seems likely that some preformed messengers are present in the mature ovum (Malkin et al., 1964; Denny and Tyler, 1964; Gli§in and Gligin, 1964). It seems also probable now that some messenger RNAs might be synthesized in the cleaving eggs after fei~ilization (see discussion in Sherbet and Lakshmi, 1965). Recent experiments of Gross et aL (1965) indicate that most of the RNA made in the cleavage phase of development has a base composition very similar to DNA and therefore might be messenger RNAs. Brachet (1965) categorically asserts that results of actinomycin and pulse experiments with p32 and Ha-ufidine conducted in his laboratory (Brachet et aL, 1963a, b) and the actinomycin experiments of Gross and Cousineau (1963, 1964); Gross et aL (1964) indicate that the nucleus does not produce large amounts of messenger RNAs immediately after fertilization. However, the recent work of Brachet and Ficq (1965) with C14-actinomycin cautions us against any naive interpretation of actinomycin experiments. They have shown that it binds with cytoplasmic DNA of amphibian oocytes. Hence a lack of susceptibility of pregastrulation embryos may not mean that messenger RNAs are not synthesized in these embryos. The pregastrulation messengers, if finally proved to be synthesized, are probably concerned with the synthesis of enzyme systems and cofactors essential for the synthesis of proteins needed for the cleavage divisions (Hultin, 1961) or those characterizing differentiation and presumably also the establishment of structural material of the cytoplasm. In other words, the genetic code seems to be transcribed in two phases. The first stage is in the pregastrulation phase where there is yet no cellular differentiation nor even signs of a stable determination of creodes. The second step is in the post-gastrulation phase. Collier (1965) suggests that determination as well as differentiation are accompanied by gene transcription and raises the question of whether the same genes are read over again or a different group of them.

IV. TIlE ROLE OF COMPETENCE IN DIFFERENTIATION It is recognized by all embryologists that the state of competence of the reacting tissue plays an important role in embryonic induction. There appears a changing pattern of competence in time. The available data indicate that there is sequential appearance as well as loss of competence during development. For

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example, there occurs a gradual restriction of potencies of different parts of the embryo (Nieuwkoop, 1952; Waddington, 1954). Holtfreter (1938) found that "aging" of gastrula ectoderm causes a change in the pattern of response to one and the same stimulus. It is clear that a change in the state of competence is iuvolved. Flickinger (1962) reviewed some data on the presence of organspecific antigens in the presumptive regions of certain organs or in the iris in cases where Wolitian regeneration of the lens is reported. According to Flickinger, though there is no correlation between the existence of antigens and the presumptive region, "these organ specific antigens may 1effect the potency of these cellular areas to form particular organs". In other words, it could be reflection of their potential competence. In some instances appearance of competence seems to be an autonomous process (Waddington, 1940). An attempt will be made later to define competence and loss of competence. Another obvious controlling factor, therefore, is the state of competence of the reacting system. If the optimum level of messenger RNAs are not synthesized before the loss of competence is complete, abnormal organogenesis might result. The pattern of malformation of organs caused by agents which interfere with the synthesis of specific RNAs will naturally depend upon the appearance of competence of the concerned reacting system.

The Nature of Competence As has already been stat~l, it is the reacting tissue which is physically moulded in the process of embryonic induction. Even if the inductor belongs to a different species, the response to the stimulus is characteristic of the reacting tissue (Spemann and Schott6, 1932; Holtfreter, 1935; Rotmann, 1935). Waddington (1940) stated: "There can be no doubt that the differences are dependent on the kind and arrangement of the genes in the two species. The available evidence shows that competence is closely connected with the processes directly controlled by the genes in that zygote." The appearance of competence could be viewed as a priming of some genes of the reacting system. As a result of interactions with the cytoplasm the regulator genes are activated which produce the allosteric represser molecules. The effector, in the form of inductive stimulus, has not yet arrived and the structural genes remain in a "repressed state". It is possible that this process itself may cause the progressive determination of the different parts of the embryo. It is worthwhile to recall the data on localization of organ specific antigens reviewed by Flickinger (1962) and which have already been discussed. This might well be a result of priming of the genes. When the concerned genes are primed the tissue can be deemed to be competent. What possibly happens in induction is a "derepression" of structural genes as a result of the combination of the inductor molecules (the effectors) with the allosteric regulator gene products and the genetic code is transcribed and the message carded to the sites of protein synthesis (see Fig. 3).

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The effector-repressor interactions could be distinctive. Any effector molecule cannot combine with a repressor molecule and make it "derepress" the structural genes normally derepressed through the agency o f the effector inductive stimulus. The effector is specific in ,he sense that only certain molecules can bring about the formation of the correct reactive sites to combine with the gene inhibitor. Similarly the repressor allosteric molecule is also in a sense specific. Since embryonic inductozs themselves are not species specific and a wide variety o f inductors such as is reported, many macromolecules seem to be capable of bringing about changes in the reactive site of the repressor molecules. The non-specificity of the inductors need not be construed as constituting any evidence against the validity of this scheme. The loss of competence of reacting tissue or the presumptive differentiating tissue is as much an important factor as the appearance of competence in the processes of differentiation. In the above scheme loss of competence can be seen as a consequence of enzymatic destruction of the repressor protein molecules. It does not seem probable that the same molecules could derepress genes o f another operon. It seems likely that there are other factors which are concomitant with the appearance of competence or even may contribute to its appearance. For example, very little is known about the specificity of ribosomes. Having known that differentiation is intricately connected with the structural organization of the cell, we shall be making the same mistake o f underestimating the role of ribosomes as did embryologists some years ago the role o f the reacting tissue in the induction phenomenon. Wright's (1963) work and his discussion (pp. 27-32) are enough evidence for the existence of different classes o f ribosomes probably having specific functions. The role of ribosomes has been discussed in a recent paper (Sherbet and Lakshmi, 1965). There seems to be every possibility that some specificity may lie at the level of the ribosomes which accompanies the process of priming of genes. Competence may thus involve two processes, viz. priming of genes and sensitization of the protein building machinery. The arrival o f the effector inductive stimulus sets the machinery into motion.

V. D I F F E R E N T I A T I O N IN GRADIENT SYSTEMS In organisms like the echinoderms distinct segregation of ooplasms is not seen, but definite morphogenetic gradients are present (Hrrstadius, 1937, 1939, 1952). If these gradients consist of distribution of effector substances, one can build up a picture of this differentiating system. Cytoplasmic and nuclear interactions must be a universal phenomenon, which, as we have suggested for other differentiating systems, cause a preliminary gene activation which is the process of priming. When genes are primed, along these morphogenetic gradients the effectors having been already present, the circuit of gene transcription is completed and this results in the production of specific messenger molecules. This suggestion is compatible with the observation (see Runnstrrm,

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1964) that these embryos show a gradient of especially ribonuclease sensitive nuclear RNAs. Further, chemicals which induce animalization induce synthesis of these RNAs. There is also very convincing experimental data that posterior parts of the embryo, i.e. those in which the vegetal-animal gradient is dominant have a moderating effect on the differentiation of the animal halves. These data lend support to our suggestion of the role of the rate of messenger synthesis. When micromeres are transplanted to animal halves the effectors present in them compete with the effectors of the animal-vegetal gradient and reduce the synthesis of messengers which are formed by the presence of the latter. This in effect regularizes the messenger gradients and brings about normal differentiation. While fully recognizing the purely hypothetical nature of the molecular basis suggested, we presume it will be easily acceptable that differentiation could be explained in terms of the two basic phenomena of epigenetics, viz. nucleocytoplasmic interactions and inductive interactions. These in their turn are resolvable into the processes of differential activity of genes causing a diffeiential synthests of messengers. Control mechanisms could exist at the level of the messenger RNAs and their rates of synthesis, and the appearance and loss of competence which constitute the priming effect ofnucleocytoplasmicinteractions.

ACKNOWLEDGEMENTS

The author is thankful to Professor J. A. V. Butler, F.R.S. for reading th© manuscript. The experimental work on the effects of FSH on differentiation was done in the University of Poona, India. This review was undertaken as part of a programme supported by grants to the Chester Beatty Research Institute, Institute of Cancer Research, Royal Cancer Hospital, from the Medical Research Council and the British Empire Cancer Campaign for Research, and by the Public Health Service Research Grant No. CA-03188-08 from the Nadonal Cancer Institute, U.S. Public Health Service.

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