The Act of Creation

by Arthur Koestler

  Even this very sketchy outline indicates the hierarchic organization of the living cell - -once considered the ultimate 'atom' of life. The genetic code is blue-printed in the chromosomes; but the chromosomes do not deal directly with the sub-matrices on lower levels of the hierarchy. They do not interfere with the stepwise operations of breaking down glucose into phosphoglycerate, into lactate, into pyruvate, into citrate, and so forth; these operations, just as those of the spindle apparatus of the centrosomes, are governed by their own sub-codes. Each organelle is a highly integrated structure and enjoys a considerable amount of functional autonomy. Its operations are switched on or off by signals from the higher echelons; but these signals are addressed, as it were, to the code which governs the action-pattern of the whole organelle, and not to its subordinate parts. Generally speaking, we shall see that a matrix on any level of the hierarchy is represented on the next-higher level by its code. Or, to put it the other way round: the members of a matrix are sub-matrices which respond as functional units to signals activating their codes.

  Nucleus and Cytoplasm

  The fertilized egg contains the total pattern of the unborn individual. This privileged position of a single cell representing the whole is of short duration: after the first few cleavages, the daughter cells begin to differentiate; they lose their potential capacity of reconstructing the whole individual, and are reduced to being parts of the growing embryo.

  The process involves both the nucleus and the cytoplasm, but in different ways. The characteristics of different types of cells, tissues, organs, are essentially the characteristics of their cytoplastic structure, which vary from type to type. The nuclei which (jointly with the cell's environment) determine that structure also differ according to cell-type, but in a subtler, more 'functional' than 'structural' way. It is generally assumed that each cell in the mature organism inherits a complete set of the genetic blue-print in the DNA chains of its chromosomes; but only a fraction of the set remains active -- i.e. those genes which govern the cell's specialized functions; the remainder is permanently 'switched off'. As mentioned before, the activity of the enzyme-producing genes is supposed to be controlled by 'operators' and 'repressors' built into the chromosomes; and these regulators in turn are controlled by feedback from the cytoplastic environment. [5] Thus the changes in the nucleus could be described as functional specialization: only certain sub-codes -- fractions of the complete code -- remain operative; whereas the changes in the cytoplasm of successive generations amount to structural individuation.

  The nuclear changes can actually be observed under the microscope. The salivary gland cells of midge larvae for instance possess large bundles of chromosomes, which are seen as sausage-like structures, with occasional swellings or puffs -- the so-called Balbiani rings. The puffs are the sites of intense RNA production; it is therefore assumed that they indicate active genes. The pattern of these swellings changes according to the age of the larva and the type of cell.*

  The action of the cytoplasm on the nucleus has also been directly demonstrated. When the nucleus from the salivary gland cell of a drosophila larva is transplanted into the cytoplasmic environment of a cell at an earlier stage of development, the chromosomes again undergo very marked changes: certain swellings disappear and others appear in their place. This clearly indicates the existence of a feedback mechanism whereby the development of the cytoplasm as a result of gene activity in its turn calls forth the activity of particular genes. [6]

  The reverse type of experiment demonstrates the action of nuclei of different ages on the same cytoplasmic environment. The nucleus of an unfertilized frog-egg is removed, and replaced by a nucleus from a developing frog-embryo. If the transplant nucleus was taken from an embryo in the early blastular stage, the result will be a clone of normal tadpoles; if it was obtained from embryos in later stages of development, it will produce abnormal forms.

  These experiments indicate not only that the nuclei undergo changes in the course of differentiation, but also that in the course of these changes they progressively lose their erstwhile 'totipotentiality'. The more the cell specializes in the role of a part, the less it is capable of creating a new whole.

  This does not mean that the DNA chains which contain the genetic code of the whole organism are lost in the differentiated cell -- it only means that as differentiation progresses, more and more genes are debarred from activity. Those which remain active are genetic sub-codes, governing the particular sub-skills which the specialized cell is called on to perform. Only the future germ-cells, segregated and protected from the beginning, retain their total creative potential to continue the genetic line -- they specialize in immortality as it were. Specialization, in morphogenesis as in other fields, exacts its price in creativity.

  The transplant experiments which I have briefly mentioned, and other evidence clearly show that while on the one hand the nuclear code governs the activities of the cell-matrix, the cytoplasm, on the other hand, is in communication with the cells' outer environment, and by feeding information on the total state of affairs back to the nucleus, co-determines which sub-code should be switched on next. The code as a whole is unalterable; but the choice of the sub-codes to be activated depends on the 'lie of the land', as in other skills. The destiny of a cell depends on the composition of the cytoplasm which it inherited from its mother cell (e.g. more animal or vegetal stuff) and on its spatial position in the growing embryo.* We have here the equivalent of a flexible strategy in morphogenesis: the development of the individual cell is determined by its invariant code and by the hazards of environment. The code represents the fixed rules of the game: if you get into the ectoderm, you must do this; if into the endoderm, you must do that. Both the fixity of the code and the flexibility of strategy become more evident as we turn to later stages of development -- the matrices of morphogenetic fields, which differentiate, in hierarchic order, into organ-systems, organs, and organ-parts.

  Regulative and Mosaic Development

  In the five-day-old salamander embryo, whose development is fairly typical for vertebrates in general, transplantation experiments make it possible to distinguish well-defined areas which will give rise to the eye, gill, limbs, kidney, etc., although not the faintest indication of these organs is as yet visible. At this stage, the tissue of a limb-area transplanted into a different position on the embryo, or on another embryo, will form a complete limb; even a heart can be formed on an embryo's flank, Such autonomous, 'self-determining' tissue-areas are called morphogenetic fields. If half of the heart, limb, or eye area is cut away, the remainder of the field will form not half an organ, but a complete heart, limb or eye -- just as, at the earlier, cleavage state, each half of a frog-egg, mechanically broken up, will form a complete frog. Moreover, if the tissue of, say, the kidney area is (by centrifuging) completely disintegrated into freely floating separate cells distributed at random, these cells, suspended in a proper medium, will in due time produce rudimentary kidneys -- just as the dissociated cells of a living sponge, which has been broken up by straining through a filter, will start to form new cell aggregates and end up by forming a complete, normal sponge. [7]

  Thus a morphogenetic field behaves 'as a unit or a whole and not merely the sum of the cellular materials of which it is composed. The field with its organizing capacities remains undisturbed if the cellular material which it controls under normal circumstances is diminished or enlarged. The unit character of the field finds its clearest manifestation in these regulative properties.' (Hamburger. [8])

  The various fields of the future organs and limbs form a mosaic in the embryo as a whole; at the same time they display remarkable regulative properties towards their own parts; -- they are again Janus-faced entities. Each organ primordium is, when 'looking upward', a member of the total matrix; when 'looking downward', a self-governing, autonomous sub-whole. Although the future of the field in its entirety is clearly predetermined on the mosaic principle, the future of its parts is still dependent
on regulative factors. The cell-populations which constitute an organ-primordium have lost their genetic totipotentiality, but they still possess a sufficient amount of multi-potentiality to keep the matrix of the field flexible. The shape of the future organ is fixed, but the part which a given cell-group or single cell will play in it is again dependent on biochemical gradients and inducers in the environment, which will trigger off the appropriate genes in the cells' genetic code.

  The differentiation of organ systems, organ parts, etc., is a stepwise affair which has been compared to the way a sculptor carves a statue out of a block of wood. With each step in development, the functions assigned to each group of cells become more precise, and more of its genetic potential is suppressed -- until in the end most cells lose even their basic freedom to divide. By the time the fertilized ovum has developed into an adult organism, the individual cell has been reduced from totipotentiality to almost nullipotentiality. It still carries the coded blue-print of the whole organism in its chromosomes, but all, except that tiny fraction of the code which regulates its specialized activities, has been permanently switched off.

  Organizers and Inducers

  The embryo grows; the adult behaves. Growth is controlled by the genetic code; adult behaviour by the nervous (and hormonal) systems.* But in between the initial and the final stage there are some transient controlling agencies at work, which catalyze development by a mechanism as yet incompletely understood: the organizers or evocators.

  During the earliest stages of development the growth of the embryo takes place in a fairly stable environment, so that feedback-controls play a relatively minor part. But with the beginning of gastrulation the situation changes: from now on each differentiating tissue acts as 'environment' on adjacent tissues; the various types of cell-population interact within the embryo.

  A particularly important cell-population originates in the grey crescent of the zygote; reappears as an analogous crescent on the blastula; gives rise to the dorsal lip, migrates into the internal cavity of the gastru]a, where it ties its place in the chordamesoderm, and becomes the so-called 'primary organizer' of the embryo, specifically concerned with initiating its nervous system -- to which it will eventually hand over control The tissue in the ectoderm which lies directly above it is destined to become the neural plate -- but only if it remains in physical contact with the organizer. If that contact is prevented, the ectoderm will not form a neural plate and there will be no nervous system. If, on the other hand, organizer tissue from the dorsal lip is grafted on to the flank of another salamander embryo which is in the process of gastrulation, it will invade the host and produce a complete Siamese twin, composed partly of the invader's tissue and partly of host tissue. It was this remarkable experiment, first performed by Spemann and Hilde Mangold in 1925, which earned the privileged region of the dorsal lip in the gastrula the name of 'primary organizer'.

  At a later stage, the organizer tissue seems to differentiate into head-, trunk-, and tail-organizers; and with the appearance of organ primordia, its inductive functions are further divided up and handed over to centres located in the organs themselves. A classic example of induction is the formation of the vertebrate eye. The rudimentary brain has two sacs, or vesicles, attached to it: the future eyes. The brain and its eye primordia originate as thickenings of the surface area which, after the in-folding of the neural tube, come to lie under the surface. So the eye vesicles must now move outward again to make contact with the surface, but at the same time remain attached to the brain by the optic stalks (which will develop into the optic nerves). In the process, the vesicles assume the shape of concave saucers, the optic cups. When these make contact with the surface, the skin areas overlaying the contact areas fold neatly into the hollow cups, thicken, detach themselves from the surface, and eventually become the transparent lenses. It can be shown that it is the optic cup which induces the skin to make a lens, for if the cup of a frog embryo is removed, no lens will form; and vice versa, if the eye vesicle is grafted under the embryo's belly, the belly skin will form a lens.

  However, the docility of embryonic tissue has its limits. The tissue must be 'competent' [9] to react to the inductor; and 'competence' is determined by the degree of differentiation the tissue has reached -- or, put in another way, by the amount of genetic multipotential which it still retains. An inductor 'cannot make any cell produce any specific response unless the cell is intrinsically prepared to do so'. [10] A given region of the ectoderm at a given stage of differentiation may retain enough genetic flexibility to become either a lens or skin-tissue; it will not be prepared to form a kidney. In the experimental laboratory, a transplanted eye-vesicle can be used to induce a lens on the salamander's belly. But under normal conditions the inductor's function is to catalyze or 'evocate' the actualization of the genetic potentials present in the appropriate tissue. Hence the term 'evocator-substance' for the chemical agent responsible for induction.

  A curious fact about inductors is that they seem to be organ-specific but not species-specific. The optic cup of a frog transplanted under a salamander embryo's skin will cause it to produce a lens; the primary organizer of the salamader will induce brain structures not only in frogs but even in fish; [11] and the organizer of a frog, even of a fish, can induce secondary embryos in the obliging salamander. But the induced embryo will be a salamander, not a frog or a fish; and the frog-skin transplanted on to the salamander's head will form a frog-mouth, not a salamander-mouth. In this respect, too the evocator seems to act merely as a trigger-releaser on the genetic potential of the cell.

  This assumption was confirmed when Holtfreter, J. Needham, and others discovered that rudimentary nervous systems could be inducted in salamander embryos by a great variety of living or dead organizers. These include most tissues of the adult salamander itself; mouse-liver and insect organs, molluscs, acidified salt solutions, sterols, and dye stuffs. Moreover, it was found that some tissues (such as embryonic skin and intestine) which cannot act as inductors when alive can do so when killed (in alcohol or by heating). All this points to the conclusion that the evocator of the nervous system is a non-specific chemical agent whose function is merely 'to release the true active substance from neighbouring cytolyzing cells'; [12] and that the substance thus released is RNA, the carrier of the cell's genetic instructions. It has indeed been shown that there exist distinct RNA gradients in the inducted tissues, and that the highest RNA concentrations are found in nervous-systems induction. Since the evocator substances, unlike hormones, act only by direct contact, i.e. by diffusion from cell to cell, it seems that their function is merely to activate those RNA sub-codes which will specify the tissues' destiny. This is in keeping with Hamburger's definition of embryonic induction as 'a process in which one developing structure, the inductor, stimulates an adjacent structure to undergo a specific differentiation'. [13] Artificial induction through transplant experiments would then amount to drastic changes in the environment of a cell-population, which interfere with its biological time-clocks (the pre-set sequence of gene-activities); just as the transplantation of a nucleus into a different cytoplasmic environment causes a change in the pattern of its chromosome-puffs.

  Induction is a transient method of regulating development, where the genetic potentials of certain cell-populations, or morphogenetic fields, are activated by chemical agencies diffused in their immediate environment. Although the chemistry of induction is still a problematic affair, it seems safe to assume, as Mittasch has pointed out, that 'organic catalyzers also show a rank order: beginning with the enzymes, which are adjusted most specifically to carry through a single reaction, to biocatalyzers such as the . . . organizer substances in animals which regulate more or less wide complexes of processes, and up to directing biocatalyzers, such as many hormones, that influence to a large extent the whole organism psychophysically.' [14]

  Past the early and transitory phase of induction, more advanced methods of co-ordination and control make their appearance. In
the human embryo the' heart begins to beat at the end of the third week, controlled by its own pace-maker, when the whole creature is less than a fifth of an inch long. Muscle contractions in response to external stimuli can be elicited after the eighth week, and spontaneous movements may begin in the tenth week. They are myogenic reactions of the muscle tissue to direct local stimulation, while the nervous system is still in the making. But the conspicuous readiness of the neural plate to start growing in response to non-specific 'evocators' designates it, as it were, as the heir apparent to the earlier forms of integration.

  To sum up: at various stages of embryonic development, and at various structural levels, we find different biochemical mechanisms, but analogue principles at work. At every stage and level the game is played according to fixed rules but with flexible strategies (although their flexibility is normally hidden from the eye and revealed only by the transplantation and grafting techniques of experimental embryology). The overall rules of the game are laid down in the complete set of instructions of the genetic code; but the particular set of instructions operative at any level at any time is triggered off by messages from the inter- and extra-cellular environment, which vary in character according to structural level and developmental stage: fertilizing agents, cytoplasmic feedbacks, direct-contact evocators, hormones, and other catalysts.