Heredity and Development: Second Edition (1972)

Below is the uncorrected machine-read text of this chapter, intended to provide our own search engines and external engines with highly rich, chapter-representative searchable text of each book. Because it is UNCORRECTED material, please consider the following text as a useful but insufficient proxy for the authoritative book pages.

HEREDITY AND DEVELOPMENT: SECOND EDITION 279 13 Developmental Control of Genetic Systems The differentiation of embryonic cells must have as its basis the differentia- tion of the cell’s genotypes. This statement must come as a surprise when one recalls a fundamental principle of genetics: mitotic cell division produces daughter cells with genetic systems identical with those of the parent cell. While this principle is probably true, the supporting data, listed below, are not absolutely convincing. 1. The data of cytology show that the chromosomes of daughter cells are identical in number, structure, and staining characteristics with those of the parent cell. 2. There are many nuclear divisions between the zygote and the adult of a species (about 35 in the frog, for example). In all the nuclear divisions of those cells in the lineage of ova or sperm, genetic integrity is main- tained. We are sure of this because the genetic systems of ova and sperm can be tested by uniting them and studying the phenotype of the new individual. Only in exceptional circumstances, can we test as rigorously the genetic systems of differentiated somatic cells. 3. In many instances at least some of the somatic cells possess the same genotype as was present in the zygote. Thus, small parts of a Hydra or of a planarian worm can regenerate an entire individual. Some of the cells, therefore, must have the complete genetic information of the species. Let us consider these facts in relation to the conclusions of Chapter 8. The data for the genetic control of protein synthesis are convincing. The DNA code is transmitted by the messenger RNA which directs the 

HEREDITY AND DEVELOPMENT: SECOND EDITION 280 synthesis of specific proteins. One message from DNA leads to the synthesis of hemoglobin; another message leads to the synthesis of insulin. This explanation of the control of protein synthesis is probably correct but some exceedingly difficult questions remain unanswered. In man and other vertebrates, hemoglobin is synthesized only in the cells that are about to become red blood cells. Similarly, insulin is synthesized only by the islet cells of the pancreas. Thus, so far as synthetic abilities are concerned, the red blood cells are differentiated one way and the islet cells another way. The differentiation of these two cell types begins in the early embryo: red blood cells arise from the mesoderm and islet cells from the endoderm. As the embryo develops, the two types of cells become increasingly different mor- phologically and finally differ in the specific proteins that they synthesize. How can one explain the origin of these two cell types? One hypothesis might be: genes for hemoglobin synthesis are present in red blood cells but are absent from islet cells; genes for insulin synthesis are present in islet cells but are absent from red blood cells. Such a hypothesis holds that there is a genetic difference between blood cells and islet cells—a difference reflected in their microscopic appearance and in their unique proteins. This hypothesis is clearly at variance with the data discussed in the first paragraph, which suggest there is an identical genetic system in all of an indi- vidual’s cells. There are two main ways of resolving the dilemma. First, we could assume that the data suggesting the genetic identity of all differentiated cells are inadequate and that the blood cells and islet cells are genetically different. Possibly only those cells that will form ova or sperm maintain intact the entire genetic system of the individual. Each type of somatic cell can then be thought of as genetically different from all other types. Evidence of the genetic nature of somatic cells is nearly always indi- rect, since normally only the genetics of germ cells can be tested adequately. Some evidence, such as regeneration in Hydra and planarian worms, suggests that at least some somatic cells have the full genetic system of the individual. Other evidence, such as the differing synthetic abilities of blood cells and islet cells, suggests that somatic cells may be genetically different. Second, we could maintain that all cells, somatic and germ, have the same genetic systems, but that not all genes are active in all cells or at all times. With this hypothesis, we would assume that islet cells possess genes respon- sible for both hemoglobin synthesis and insulin synthesis, but that only those concerned with insulin synthesis are active. The genes 

HEREDITY AND DEVELOPMENT: SECOND EDITION 281 controlling hemoglobin synthesis would be inhibited in these cells. Further- more, we might think of this inhibition as being either irreversible or reversible. If irreversible, it would be impossible for the insulin-synthesizing cells of the islets to produce hemoglobin under any experimental conditions. If, on the other hand, the inhibition could be removed, hemoglobin might be produced. There are many data that bear on these possibilities, but not enough for us to reach unequivocal conclusions. Some of these will now be considered. Tests of Genetic Identity of Somatic Cells. Since the days of Roux, every student of development has been interested in the nature of the genetic sys- tem of somatic cells. Spemann had been able to show that a single nucleus from a salamander embryo in the 16-cell stage, plus some cytoplasm, was able to develop into an entire embryo. Technical problems prevented his test- ing the developmental potentialities of older nuclei. In 1952 Robert Briggs and Thomas J.King (then of the Institute for Cancer Research in Philadelphia) perfected a method for testing the nuclei of blas- tula and even older embryonic stages (Fig. 13–1). Basically 13–1 The Briggs and King method of transferring nuclei. A piece of an older donor embryo, in this case the roof of the blastocoel of a blastula, is removed and placed in a solution that causes the cells to fall free of one another. One cell is drawn into a micropipette and then injected into the host embryo. The host was previously prepared as follows: first, it was activated by being pricked with a glass needle; second, the egg nucleus was removed by flicking it out with a glass needle. 

HEREDITY AND DEVELOPMENT: SECOND EDITION 282 their method consists of transferring a nucleus from older embryos to an unfertilized ovum from which the egg nucleus has been removed. The nucleus to be transferred is obtained as follows. In their first experiments, Briggs and King used embryos in the middle blastula stage. A portion of the blastocoel roof was cut off and a single cell from its underside drawn into a micropipette. The pipette has a bore smaller than the diameter of the cell. Thus the cell membrane is broken as it enters the pipette but the nucleus remains intact. The broken cell with its intact nucleus is then injected into the enucleated ovum. The injected ovum may divide and form an embryo. The experiment is difficult for both the experimenter and the embryo. Not infrequently the latter is injured, and either dies or develops abnormally. Other embryos, however, develop normally, and from these one can con- clude that the transferred nucleus possesses all the information required for normal development. The injected nucleus of such an embryo would seem to be genetically identical to the zygote nucleus. Nuclei from cells of various parts of the blastula were tested in this manner. Briggs and King could find no evidence of any nuclear differentiation at this early stage. Subsequently, John Gurdon of Oxford University was able to obtain nor- mal embryos when the injected nucleus came from a larva, in which the cells were fully differentiated, or even from nuclei of adult cells. These discover- ies gave added support to the hypothesis that all of an individual’s nuclei are genetically identical. This made it all the more difficult to understand how embryonic differentation can come about. One has to accept these facts: 1. The experiments of Briggs and King and of Gurdon seem to demon- strate that the genes of all cells in an individual are identical. 2. Since different sorts of cells produce different sorts of proteins, their genes must be functionally different. Non-nuclear Control of Gene Action. A hypothesis is available that unites these apparently contradictory facts. One can assume that the same genes are present in all cells but that not all function in the same way. Differentiation could be a consequence of differential activation or suppression of genes. The basic mechanisms could be those discovered in microorganisms, as summarized in Figure 8–9. Recall that whether or not a gene in E. coli is active seems to depend on the biochemical environment in the cell. There is a large amount of embryological data to suggest that factors exter- nal to the genes will determine which ones are active and which ones are not —and hence these external factors will control the direction 

HEREDITY AND DEVELOPMENT: SECOND EDITION 283 of differentiation. Some of the evidence that supports this hypothesis will now be cited. Most geneticists of the first third of the twentieth century probably looked upon genes as controlling the cell’s activities in ways quite uninfluenced by the cell as a whole. To be sure, genes required the substance of the cytoplasm for their work but this substance was something to be molded by the genes, not something to mold them. The data of genetics were most readily inter- preted in this manner. Such an interpretation, however, was quite unaccept- able to experimental embryologists. They were impressed by the importance of cytoplasmic materials, especially in mosaic eggs. One of the most dramatic examples was discovered in the mollusk, Dental- ium, by E.B.Wilson, whom we met as a cytologist many chapters back. The pattern of cleavage in Dentalium is precise and each cell has its fixed role in development. The first event that one notices after fertilization is the forma- tion of a protuberance near the vegetal pole (Fig. 13–2), called the polar lobe. (Similar s tructures are found in many invertebrates but neither their function nor significance has ever been fully determined.) The plane of first cleavage goes to one side of the first polar lobe and, when cleavage is complete, the polar lobe flows into one of the two cells. The result is a smaller cell, known as AB, and a larger one with the contents of the polar lobe, known as CD. Shortly before second cleavage, the CD cell forms a second polar lobe. Its contents flow back into one of the cells, known as D at the end of the second cleavage. Third cleavage divides the embryo into four slightly smaller animal cells, known as 1a, 1b, 1c, and 1d, and four slightly larger vegetal cells known as 1A, 1B, 1C, and 1D. 13–2 Dentalium. The pattern of cleavage in the early embryo and the trochophore larva. 

HEREDITY AND DEVELOPMENT: SECOND EDITION 284 The cleavages continue in a very exact way and after a day a trochophore larva is formed. This is a spindle-shaped little creature with a tuft of long flagella at the apical end and a band of cilia, the prototroch, around its middle. Wilson found that it is possible to separate the cells of the early embryo and he did so in the hope of finding the role that each plays in the formation of the trochophore larva. The first thing he did was to cut off the first polar lobe. Apart from the fact that the second polar lobe failed to form, early development seemed to be normal. The larva, however, was a disaster. It lacked an apical tuft and the entire region posterior to the prototroch. Clearly the first polar lobe is essen- tial for the formation of a normal trochophore. After first cleavage occurred, he isolated the AB and CD cells. The AB cell failed to form a second polar lobe but otherwise it cleaved as though it was still part of a whole embryo (i.e. mosaic development). It formed a larva that lacked both apical tuft and posttrochal region. The CD did form a second polar lobe and cleaved as though part of the whole. It produced a larva with both the apical tuft and the posttrochal region (an unusually large one, in fact). Thus, something necessary for the formation of the apical tuft and the post- trochal region is localized in the first polar lobe and then passes into the CD cell. Wilson removed the second polar lobe and observed that the larva still lacked the posttrochal region but almost always had an apical tuft. When the individual cells of the four-cell stage were isolated, A, B, and C each produced a larva lacking the apical tuft and the posttrochal region. D alone produced a larva with both. Finally after the third cleavage had occurred, he isolated the four cells of the animal hemisphere: 1a, 1b, 1c, and 1d. All produced swimming larvae. None had a posttrochal region and 1d alone had an apical tuft. Putting these data together, Wilson concluded that the substances in the egg that are necessary if the posttrochal region is to develop are originally in the vegetal hemisphere of the uncleaved egg. Subsequently they are localized in the first polar lobe, CD cell, second polar lobe, and D cell. Similarly, the materials necessary for the apical tuft are first in the vegetal hemisphere and then in the first polar lobe, CD cell, D cell, and then in the 1d cell. The polar lobes do not contain a nucleus so the substances responsible for the apical tuft and the posttrochal region must be cytoplasmic. Without these substances the genes of Dentalium are unable to make either 

HEREDITY AND DEVELOPMENT: SECOND EDITION 285 an apical tuft or the posttrochal region. When these substances are present, they can. If the model for gene action that was proposed for E. coli (Fig. 8–9), holds here as well, possibly the observations on Dentalium can be interpreted as follows. The genes that are involved in the formation of the apical tuft are normally ‘turned off.’ That is, their regulator proteins are combined with the operator sites and prevent the synthesis of mRNA. The substances that were in the first polar lobe and finally in the 1d cell are able to combine with the regulator proteins and prevent them from attaching to the operator sites. With no repression, the genes can make the mRNAs that are responsible for the formation of the apical tuft. This hypothesis is consistent with the thinking of embryologists, who fail to see how a genetic system, identical in all cells, alone provides for cellular differentiation. They conceive, instead, that external conditions or cytoplas- mic substances interact with a uniform cellular genetic system to provide for differentiation. Though the genetic system specifies what a cell may do, non- genetic phenomena influence what it actually does. This point of view, which once would have been reasonable to an embryologist but not to a geneticist, now seems reasonable to both. Now another example will be given. Cytodifferentiation in Fucus. Cellular differentiation in the seaweed Fucus (commonly called rockweed) is present at the first possible opportu- nity, that is, by the two-cell stage. At first the zygote is spherical (Fig. 13–3). Before first cleavage begins, a protuberance appears on one side, giving the zygote roughly the shape of a snowshoe. The polarity thus developed is the basis of further differentiation. First cleavage cuts across the long axis of the zygote, producing two cells of different shapes—only one having the protuberance. The developmental fates of the two cells are entirely different. The cell with the protuberance divides repeatedly to give rise to the rhizoid, which attaches the Fucus to the rocks. The other cell gives rise to the thallus, which is the leaf-like part of the plant. According to D.M.Whitaker, who made these observations on Fucus, ‘when the point of origin of the rhizoid protuberance is determined, the polar- ity and whole developmental pattern of the embryo is determined.’ The factors responsible for the formation of the protuberance, therefore, are of fundamental importance in determining the differentiation of cells in Fucus. Whittaker found that seemingly minor environmental differences could determine the point of outgrowth of the protuberance. If the zygotes are in a group, the protuberances are directed inward 

HEREDITY AND DEVELOPMENT: SECOND EDITION 286 13–3 Early development of Fucus and some of the factors that influence the formation of the protuberance. (Fig. 13–3). If the zygote is placed between two pipettes, one with seawater agar at pH 7.8 and the other at pH 6.4, the protuberance appears on the side with the lower pH. If the zygotes are exposed to white light, the protuberance appears on the dark side. If the zygotes are kept in a temperature gradient, the protuberance appears on the warmer side. Thus an initial stimulus, which has nothing to do with the nucleus, controls the beginning of a series of events that is of profound importance in cellular differentiation. The protuberance begins when the embryo contains a single nucleus. The first divisions occurs some hours after the protuberance has been induced, and in a plane related to the protuberance itself. Recall also that, once the protuberance has formed, the basic polarity of the developing embryo has been determined. What the nucleus will do later, therefore, is the result of an initial environmental stimulus. There is much evidence from many other organisms suggesting that the basic polarity of an egg, hence of the embryo itself, is determined 

HEREDITY AND DEVELOPMENT: SECOND EDITION 287 by conditions external to the maturing egg cell. In these important events, the egg’s nucleus seems to play no determining role. In many mosaic embryos the pattern of development is closely correlated with the distribution, during cleavage, of visibly different cytoplasmic regions. The establishment of some polarity or regional differences in an embryo is an exceedingly important factor in development. Consider the result if this were not the case: a nucleus dividing by mitosis in an unorganized cytoplasm would produce only a group of similar cells, certainly not a differentiated embryo. There must be some initial induction of a difference, some defining of polarity. Once this has been brought about, one can suggest models for the manner in which cellular differentiation can proceed, even with nuclei that are at first functionally identical. Cellular differentiation may be thought of as an interaction between nuclei, which are genetically identical, and different cytoplasmic regions. In many cases these cytoplasmic regions are formed under the control of genes that acted much earlier, usually when the ova were being formed. In other cases, such as Fucus, the environment may be important in inducing cyto- plasmic differences. Possibly the substances that give the cytoplasm its regional specificity interact with the substances (Fig. 8–9) produced by the regulator genes. Whatever the mechanism may be, it seems probable that there is a functional nuclear differentiation concomitant with the differentia- tion of the cell as a whole. A nucleus of an islet cell does not develop the way it does because of some innate specificity. Instead it is developed— developed in a specific way because of the cytoplasm in which it happens to lie. A generation ago few embryologists or geneticists would have predicted that a synthesis of their fields would be made possible by studies on the bac- terium Escherichia coli. But this microscopic creature, with no embryology of its own, has shown a way. Now it is difficult to distinguish between a geneticist and an embryologist, as they advance their science beyond what each might independently achieve. Suggested Readings Additional references will be found in Chapter 9 of the Readings. BARTH, LUCENA JAEGER. 1964. Development. Selected Topics. Reading, Mass.: Addi- son-Wesley. BALINSKY, B.I. 1970. An Introduction to Embryology. Third Edition. Philadelphia: W.B. Saunders. DAVIDSON, ERIC A. 1968. Gene Activity in Early Development. New York: Academic Press. 

HEREDITY AND DEVELOPMENT: SECOND EDITION 288 GURDON, J.B., and H.R.WOODLAND. 1968. ‘The cytoplasmic control of nuclear activ- ity in animal development.’ Biological Reviews 43:233–67. WADDINGTON, C.H. 1966. Principles of Development and Differentiation. New York: Macmillan. WHITAKER, D.M. 1940. ‘Physical factors of growth.’ Growth Supplement, 1940: 75–90. WILLIER, BENJAMIN H., PAUL A.WEISS, and VIKTOR HAMBURGER. 1955. Analy- sis of Development. Philadelphia: W.B.Saunders. WILSON, EDMUND B. 1904. ‘Experimental studies on germinal localization. I. The germ-regions in the egg of Dentalium.’ Journal of Experimental Zoology 1:1–72.