Molecular Biology of the Cell - Volume 3 - Alberts B., Bray D., Lewis J., Raff M., Roberts K., Watson J. 1994

From Cells to Multicellular Organisms
Cellular Mechanisms of Development
Cellular Memory and the Generation of Cell Diversity

A fertilized egg can develop into a male or a female, a sea urchin, a frog, or a human being. The developmental outcome is dictated by The Genome: the linear sequence of A, G, C, and T NUCLEOTIDES in the Organism's DNA must direct The formation of numerous chemically distinct Cell types, organized spatially in a specific manner. The task of developmental biology is to explain this process. First and foremost, we must answer how differences arise during Embryogenesis between initially identical Cells. To illustrate the fundamental laws of development, we will first turn to amphibians and then to mammals.

16.2.1. The genome remains constant during development, but Gene Expression changes [15]

Cells of different types differ from one another primarily because, In addition to the numerous Proteins required by all of them without exception to maintain viability, cells of each type synthesize their own unique set of specialized proteins. For example, epidermal cells synthesize keratin, erythrocytes synthesize Hemoglobin, intestinal cells synthesize digestive Enzymes, lens cells synthesize crystallins, and so on. Since cells of each type are characterized by specific sets of gene products, one might ask: is this simply explained by the fact that cells possess different sets of genes? Lens cells, for instance, might have lost the genes for keratin, hemoglobin, etc., while retaining the crystallin genes, or they might have selectively increased the copy number of crystallin genes through Amplification. However, a wealth of evidence shows that this is not the case: cells of almost every type contain the same complete genome that was initially present in the fertilized egg. The reason for differences in cell properties lies not in possessing different sets of genes, but in their differential expression. In other words, gene activity is regulated: genes can be switched on and off (see Chapter 10).

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Fig. 16-18. Diagram of an experiment demonstrating that The Nucleus of a differentiated frog Skin cell contains all the genetic material necessary to support The Development of a normal tadpole. (J. B. Gurdon, Gene Expression During Cell Differentiation. Oxford, U.K., Oxford University Press, 1973; with modifications.)

The most compelling evidence that, despite visible cellular changes during differentiation, the genome itself remains unchanged was obtained from nuclear transplantation experiments in amphibian eggs (Fig. 16-18). Typically, the size of amphibian eggs makes it possible to use a Glass micropipette to inject nuclei obtained from other cells. The egg's own nucleus is first destroyed by ultraviolet irradiation. The micropipette puncture stimulates the egg to initiate development (see Section 15.4.3). This approach makes it possible to determine whether the nucleus of a differentiated somatic cell contains a complete genome equivalent to that of normal fertilized eggs and capable of supporting development. The answer turned out to be affirmative: replacing an egg nucleus with the nucleus of a keratinocyte from the skin of an adult frog or an erythrocyte yielded normal swimming tadpoles. Such experiments have A number of limitations: they are successful only when using nuclei from certain differentiated cells and eggs of specific species. Nevertheless, results from other studies lead to the Conclusion that genomic constancy is preserved during development.

There are several known exceptions to this rule. For example, in some invertebrates, part of the Chromosomes present in the germ line cells (gamete precursors) is lost in somatic (non-germ) cells during early Selection/3.html">Stages of development. In the oocytes of certain other animals (including Xenopus laevis), selective Replication of ribosomal RNA genes occurs, while in the larvae of some insects, unequal polytenization of chromosomes takes place, resulting in the enhanced amplification of certain specific genes (see Section 9.2.6). The synthesis of Antibodies and antigen-specific receptors by lymphocytes in vertebrates involves the splicing of DNA fragments located in different Regions of the genome within these specialized cells. This splicing occurs as these cells differentiate (see Chapter 18).

16.2.2. Differences between blastomeres are often the result of an Asymmetry inherent in the egg (except in mammals) [16]

When the nucleus of an undifferentiated cell is transplanted into an enucleated Xenopus egg, the pattern of gene expression changes, and the nucleus begins to resemble a normal egg nucleus. Consequently, nuclear behavior can be controlled by its cytoplasmic environment. The egg of Xenopus and many other species is chemically asymmetric; that is, the concentration of certain components varies across different regions of the Cytoplasm. As a result, from the very beginning of development, the resulting embryonic cells differ in certain properties because their cytoplasm inherits unequal amounts of these pre-synthesized substances. Structure/19.html">The Importance of localized egg determinants varies among species. For instance, mammalian eggs are symmetrical, and therefore all early blastomeres are completely identical. At the same time, the well-known phenomenon of "mosaic" eggs occurs in Mollusks, ascidians, marine nematodes (see Section 13.5.17), and some other animal groups. Such eggs contain a specific set of localized determinants. The planes of early Cleavage divisions are oriented According to the arrangement of specific substances within the egg, and thus each blastomere inherits a predictable set of molecules (Fig. 16-19). Following the Separation of early blastomeres, which in this case develop in isolation from one another, most such isolated blastomeres will give rise precisely to those cell types that would have formed from them in a normal embryo. This controlling function of the egg cytoplasm can be demonstrated by artificially altering the distribution of egg substances relative to the cleavage planes. Such experiments can be performed on the eggs of the ascidian Styela (Fig. 16-19), where different regions of the cytoplasm are easily distinguishable because they contain different pigments. By disturbing the distribution of these substances in the embryo, one can easily predict the developmental pathway of a given blastomere based on the type of cytoplasm it inherited.

Fig. 16-19. Three successive stages of early development in the ascidian Styela. On the right is an image of an early gastrula (viewed from below), showing the forming gut cavity. Different regions of the egg cytoplasm are pigmented differently. This pigmentation is permanent and correlates with a specific pattern of cleavage divisions. The Fate of each blastomere can be predicted based on the portion of the egg cytoplasm its ancestors inherited (see also Fig. 16-29). (P. P. Grasse, Traité de Zoologie. Paris, Masson, 1966.)

The Study of mosaic eggs raises an intriguing question for cell biology: how are the pattern of cleavage and the distribution of chemical substances so precisely correlated? It is possible that both are determined by the architecture of the Cytoskeleton. Some evidence suggests that localized chemical determinants are linked to the cytoskeleton, and that the cytoskeleton itself controls the orientation of cleavage planes (see Section 13.5.13).

16.2.3. Chemical interactions between blastomeres give rise to new cell types with a more detailed spatial arrangement: mesoderm induction in Xenopus [17]

The Xenopus egg represents a sort of middle ground between the extremes exemplified by mosaic eggs and mammalian eggs. The asymmetric arrangement of components in the Xenopus egg cytoplasm causes differences between the blastomeres of the animal and vegetal poles, which can be considered a defining characteristic of the early Spatial Organization of the embryo. The Generation of the full Complement of cell types is determined by interactions among blastomeres. If early Xenopus embryos are placed in a medium lacking Ca²⁺ or Mg²⁺ ions, the blastomeres lose their adhesiveness and easily separate from one another, with each isolated blastomere capable of developing independently. Under these conditions, some blastomeres will acquire features characteristic of the ectoderm, others will develop endodermal traits, but the expression of Actin genes (specific to Muscle), which serve as a marker of mesodermal differentiation, will not be observed. The reciprocal experiment confirms that the acquisition of a mesodermal fate by cells is determined (at least in part) by intercellular interactions: when animal pole cells of a blastula are placed near vegetal pole cells, the former switch from an ectodermal to a mesodermal pathway of development (Fig. 16-20). The switching of developmental pathways under METABOLISM/18.html">The Influence of a neighboring group of cells is called induction; during normal development, inductive interactions can occur both between cells that are initially adjacent (mesoderm induction) and between cells that are brought together by morphogenetic movements, such as during Gastrulation. The interaction of multiple cell types, realized through a series of successive inductions, makes it possible to generate a vast array of diverse cell types (Fig. 16-21).

Fig. 16-20. Mesoderm induction in Xenopus. Animal pole Cells of the blastula, which normally form only ectoderm, participate in the Formation of the vegetal mesoderm when cultured together with vegetal pole cells. During normal development, such inductive interactions occur at an earlier stage; by this time, the equatorial region of the blastula has already acquired the capacity to form mesoderm when isolated in culture.

Fig. 16-21. Through a series of inductive interactions, a multitude of distinct cell types can arise from a few initial ones.

During mesoderm induction in Xenopus, direct contact between cells is not strictly required; they can be separated by some distance. This implies that the inducing agent is a diffusing substance. It turned out that fibroblast growth factor (FGF) can be used instead of vegetal hemisphere blastomeres (see Table 13-1) to induce animal pole cells to develop along the mesodermal pathway; mesoderm formation can also be induced by transforming growth factor beta 2 (TGF-β2), one of the two TGF-β variants (see Table 13-1). The action of FGF typically promotes the development of ventral mesodermal derivatives (such as Blood Cells), whereas TGF-β2 predominantly induces dorsal derivatives (such as muscle). Normal Xenopus embryos contain mRNA encoding FGF. Furthermore, mRNA for the Vgl protein, which shares partial Homology with TGF-β, is not only present in the egg but has been shown to be localized in the vegetal pole region of the egg and early embryos (Fig. 16-22). These observations indicate that FGF, TGF-β2, or related molecules mediate mesoderm induction. This and other rapidly accumulating Examples lead to the conclusion that a few dozen proteins known as growth factors play a crucial role in regulating developmental pathways. In adult organisms, these same factors regulate Cell Division and differentiation, as well as tissue repair (see Chapter 17). Much like Neurotransmitters in The Nervous system, these factors appear to be deployed in various contexts to transmit different signals between cells.

Fig. 16-22. Stained dots indicate the localization of mRNA encoding the Vgl protein in the vegetal hemisphere of a Xenopus egg at various stages of development, as demonstrated by in situ Hybridization. The Vgl protein is partially homologous to the growth factor TGF-β and may be a component of the mesoderm-inducing signal produced by vegetal pole cells at an early stage of embryonic development. The mechanism controlling the relocalization of Vgl mRNA remains unstudied.

16.2.4. Mammalian embryos develop within the protective environment of the Uterus [18]

Mammalian development has a number of unique features. The most important of these is that it takes place within the uterus, which protects the embryo and relieves it of the need to develop rapidly. In addition, because the Placenta supplies the embryo with nutrients from the maternal organism, the mammalian egg does not require large reserves of these substances in the form of yolk. Consequently, the diameter of a mouse egg is about 80 µm, making it approximately 2,000 times smaller in volume than a typical amphibian egg. Cleavage divisions are no faster than those of ordinary somatic cells, and Transcription begins as early as the 2-cell embryo stage. Later stages of mammalian development broadly resemble those of other vertebrates, such as Xenopus. However, during mammalian development, There is a major "detour" aimed at creating complex structures—primarily the amnion and placenta—which enclose the embryo proper, protect it, and provide for metabolic exchange with the mother. These structures, like all other Organs, originate from the fertilized egg, but they are called extraembryonic because they are discarded at birth and do not contribute to the body of the adult organism.

The stages of early mouse development are illustrated in Fig. 16-23. Initially, the egg is covered by a glycoprotein coat called the zona pellucida. The fertilized egg cleaves within this shell to form a morula, a cluster of cells resembling a raspberry (Fig. 16-23). As the embryo transitions from the 8-cell to the 16-cell stage, The surface of the morula becomes smoother and its shape more rounded as changes in mutual Cell Adhesion cause the cells to pack more tightly together (Fig. 16-24). Tight junctions form between the cells of the outer layer, thereby isolating the inner regions of the morula from the external environment. Subsequently, internal intercellular spaces expand to form the blastocoel, a fluid-filled cavity. The morula transforms into a blastocyst. The cells surrounding the blastocoel form a spherical vesicle at this stage, with a denser cluster of cells located at one of its poles. The outer layer of cells is called the trophectoderm, and the cluster of cells inside the blastocyst at one of the poles is called the inner cell mass (Fig. 16-23).

Fig. 16-23. Cytology/cytology/16.html">Early stages of mouse development. (Photographs courtesy of Patricia Calarco, from G. Martin, Science, 209, 768-776, 1980. Copyright 1980, American Association for the Advancement of Science.)

Fig. 16-24. Mouse embryo at early stages of development. Scanning electron micrographs with the zona pellucida removed. A. Two-cell stage. B. Four-cell stage (along with four blastomeres, a polar body is visible; see Section 15.3.3). C. Morula stage (8–16 cells); embryonic compaction is underway. D. Blastocyst. (Courtesy of Patricia Calarco; D — from R. Calarco, S.J. Epstein, Dev. Biol., 32, 208-213, 1973.)

The embryo proper forms exclusively from the inner cell mass. The trophectoderm gives rise to the placenta and forms earlier than other extraembryonic structures. Following the disappearance of the zona pellucida, trophectoderm cells come into contact with the uterine wall, into which the embryo implants. Meanwhile, the inner cell mass grows and begins to differentiate: a portion of it also forms extraembryonic structures, such as the yolk sac, while another portion forms the embryo proper. Here, much like the corresponding stages of development in other vertebrate species, processes of gastrulation, neurulation, etc., also take place, although in some cases this homology is far less visually obvious due to the specific geometry of mammalian embryos.

16.2.5. Differentiation of cells in the early mammalian embryo depends on cell-cell interactions [19, 20]

Up to the 8-cell stage, the early mammalian embryo exhibits a remarkable capacity for regulative development, with each of its cells retaining the potential to subsequently form any part of a later embryo or even an adult organism. A clear example of this is the formation of identical twins from a single fertilized egg. In this case, two fully normal individuals arise, each formed from a portion of a normal embryo. If, for instance, one of the cells of a 2-cell mouse embryo is destroyed with a needle and the remaining incomplete embryo is implanted into the uterus for further development, a completely normal mouse will be born in a large proportion of cases.

At the same time, two 8-cell mouse embryos can be combined into a single giant morula, which develops into a normal-sized mouse (Fig. 16-25). Animals arising from the development of aggregates of genetically distinct cells are called chimeras. Chimeras can also be produced by injecting cells from early embryos into blastocysts of a different genotype. The introduced foreign cells are incorporated into the inner cell mass of the recipient embryo, resulting in a chimeric animal. Chimeras can even be obtained after injecting a single cell; this makes it possible to determine the extent to which a given cell retains its developmental potential. An important conclusion follows from such experiments: cells of very early mammalian embryos (up to the 8-cell stage) are identical and possess unlimited potential, meaning they are totipotent.

Cells become different as a result of interacting with one another. In the mouse embryo, the initial differences between the cells of the inner cell mass and the trophectoderm are determined by the spatial arrangement of intercellular contacts. At the 8-cell stage, all blastomeres occupy approximately the same position; their inner surfaces contact other blastomeres, while their outer surfaces face outward. Each blastomere exhibits polarity, characterized by the presence of microvilli on the outer surface and an asymmetric distribution of intracellular components. In vitro experiments on blastomeres demonstrate that this polarity is determined by The Nature of intercellular contacts: the assembly of microvilli and associated components occurs only if that patch of the surface lacks contact with other blastomeres. The plane of the next division is oriented so as to exploit this asymmetry to produce two distinct descendants: one (the inner cell mass cell) faces inward, lacking microvilli-associated components, while the other (the trophectoderm cell) faces outward and inherits those components. Thus, the arrangement of intercellular contacts likely controls The Emergence of the first cellular differences (Fig. 16-26).

16.2.6. Teratocarcinoma cell behavior demonstrates the importance of environmental signals [21]

During early mammalian development, the fate of each individual cell depends on interactions with its neighbors, and such an embryo can be classified as a regulative system. The experiments on mice described earlier serve as a good illustration of this: cells of halved or doubled chimeric embryos adjust their behavior so that the resulting animal does not differ from the norm in either size or structure. However, under severely abnormal developmental conditions, embryonic cells escape this control. Several important Conclusions follow from this.

Fig. 16-25. Method for producing chimeric mice by fusing two morulae with different genotypes.

Fig. 16-26. A. Polarization of mouse blastomeres at the 8-cell stage results in outer and inner cells differing in chemical properties by the 16-cell stage. B. Two blastomeres from a mouse embryo at the end of the 8-cell stage, labeled with fluorescent antibodies against clathrin (left) or fluorescent concanavalin A (right). The figure shows that polarization of intracellular components as well as cell Surface Properties takes place. (B — from V. Maro, M.H. Johnson, S.J. Pickering, D. Louvard, J. Embryol. Exp. Morphol., 90, 287-309, 1985.)

When normal early embryos are transplanted into the Kidneys or Testes of adult animals, embryonic organization is disrupted and normal control over cell proliferation is compromised. This abnormal growth results in a teratoma — a disorganized mass of cells containing various differentiated Tissues (skin, bone, Glandular Epithelium, etc.) intermingled with undifferentiated stem cells that continue to divide and generate these differentiated tissues. A teratoma can also arise spontaneously as a result of an accidental developmental mishap. Under normal conditions, cleavage and embryogenesis begin only after the egg has been fertilized by a sperm. This event serves as the trigger for development. However, in some cases, an oocyte initiates development spontaneously. This spontaneous (Fertilization-free) development is termed parthenogenesis (see Section 15.4.3). If this occurs before the oocyte is released from the Ovary, the embryo develops almost normally up to the blastocyst stage, after which a teratoma forms. Teratomas can also arise in males from primordial Germ Cells within the testes; in rodents, teratoma formation can be artificially induced by transplanting developing embryonic testes containing primordial germ cells into an adult animal.

In all these cases, highly similar teratomas arise, and all of them can be used to derive transplantable malignant tumors known as teratocarcinomas. A teratocarcinoma is capable of growing until it causes the death of the host. By transplanting Cancer cells from one individual to another, a teratocarcinoma can be maintained indefinitely. A teratocarcinoma contains a population of undifferentiated stem cells alongside differentiated cells derived from those stem cells. Teratocarcinoma stem cells can be cultured in vitro as permanent cell lines. In a suitable medium, they continue to proliferate indefinitely without differentiating. However, if the medium is changed by adding a differentiation inducer, such as retinoic acid, or if conditions are created that cause the cells to aggregate, the stem cells receive a cue to differentiate and give rise to a multitude of normal, specialized cell types.

Fig. 16-27. An experiment demonstrating that combining teratocarcinoma cells with cells of a normal blastocyst can yield a healthy chimeric mouse.

One might suppose that the intriguing properties of teratocarcinoma stem cells, much like those in other tumor types, are the consequence of Mutations in genes responsible for normal cell behavior control (see Section 21.1.2). However, further observations have shown this not to be the case. It turned out that very similar cells can be obtained by placing cells of the inner cell mass into a culture medium and, as soon as they begin to divide, dispersing them. Some of the dispersed cells continue to divide indefinitely without altering their properties; they can be used to establish permanent clones possessing many of the characteristics of normal inner cell mass cells. These cell lines are nearly indistinguishable from those derived from teratocarcinomas, but they can be obtained from normal embryos with a frequency that refutes the hypothesis of a mutational origin. Presumably, dispersing the cells and depriving them of their normal microenvironment prevents them from receiving signals that normally restrict their proliferation and promote sequential differentiation.

Moreover, the abnormal behavior of such stem cells (regardless of whether they originated from a teratocarcinoma or as a normal embryonic cell culture) can be altered by placing them back into a normal environment. To achieve this, the cells are injected into the cavity of a normal blastocyst (Fig. 16-27). The injected cells incorporate into the inner cell mass of the recipient blastocyst and participate in the formation of a superficially normal chimeric mouse: descendants of the injected Embryonic Stem Cells can be found in virtually all tissues of this animal, where they differentiate in strict accordance with their Location and are even capable of participating in the formation of viable germ cells. These experiments provide compelling evidence that signals received by early embryonic cells from neighboring blastomeres play a critical role in determining their developmental fate.

16.2.7. The behavior of multicellular animal cells is determined not only by their genome and environment, but also by their history

During organismal development, specific groups of cells must not only acquire certain characteristic features that distinguish them from one another, but they must also preserve these acquired differences even after the external signals that initiated the changes have disappeared, and pass these traits on to their progeny. Thus, a pigment cell must give rise to pigment cells upon division, a Liver cell must produce liver cells, and so on. The distinctive traits of cells arise as a consequence of the various influences they experienced during embryogenesis; the maintenance of these differences relies on the ability of cells to somehow 'lock in' The impact of these past events and transmit them to their descendants. Even the most primitive Bacteria can rapidly alter their chemical activity in response to environmental changes. However, the cells of higher animals are significantly more complex; their behavior is governed not only by their genome and present environment, but also by their past history.

16.2.8. The future specialization of cells is determined long before the appearance of external signs of differentiation [14, 22]

The most renowned proof of cellular memory is the stable Maintenance of the differentiated state in adult cells (see Section 13.4.1). Thanks to cellular memory, the stimulus that directed a cell down a particular differentiation pathway can continue to exert its effects on its descendants. Certain cells of vertebrate somites specialize as muscle cell precursors at very early stages of development and migrate from the somites to various PARTS OF THE body, including the limb-forming regions; all of this complex behavior is determined by a series of decisions made by the cells much earlier, specifically before and during gastrulation (see Section 16.6.5). These precursor cells do not yet contain large amounts of the contractile proteins characteristic of mature muscle fibers; nor do they visually differ from other cells of the limb bud that have a different origin. Only days later do they acquire the external features typical of differentiated muscle cells and begin actively synthesizing proteins specific to those cells. The remaining cells of the prospective limb located nearby differentiate into Connective Tissue elements. Consequently, the choice of the developmental program (i.e., whether a cell will become a muscle or CONNECTIVE TISSUE CELL) occurs long before any external signs of differentiation manifest. This program is presumably encoded within the cells as subtle chemical modifications (in this case, apparently involving the Activation of a primary muscle-specific regulatory gene — see Section 10.1.8).

Cells that have committed to a particular developmental program are referred to as determined. In Embryology, 'determination' is such a crucial and nuanced concept that we must give it a more rigorous definition. A cell is considered determined if a stable internal change has occurred within it that distinguishes it and its progeny from other embryonic cells and predetermines its development along a specialized pathway.

Let us examine several points of this definition.

1. The modification must make The Cell and its descendants distinct from other cells and direct these cells along a specific developmental pathway: a cell is not considered determined simply because it outpaces other cells during maturation. Determination involves the choice of a specific developmental pathway.

2. The changes occurring in differentiating Cells must be self-sustaining. Determination implies the establishment of differences that are inherited across generations of cells. A cell is not considered determined solely on The basis of occupying a particular position in the animal's body. To be determined, it must retain its characteristic features even after the disappearance of the external signals that triggered their appearance.

Determination is sometimes defined as an irreversible change. However, since we cannot test a cell's behavior under all possible environmental conditions, the reversibility of such a change cannot be completely ruled out; moreover, in some cases, cellular memory may be disrupted, rendering the determined state reversible. Therefore, we strive to avoid extreme expressions and speak of determination as a self-sustaining change in cellular properties.

The term differentiation is usually used to define overt specialization, i.e., cellular properties that are clearly pronounced. Typically, a cell becomes determined before differentiation, although in some cases both processes occur simultaneously. In principle, however, differentiation can occur without determination if the overt specialization of the cell is reversible.

16.2.9. The timing of cell determination can be determined in transplantation experiments [23]

To prove that a cell or a group of cells is determined, one must identify a specific trait in them that persists even when the conditions under which it arose are experimentally altered. In this case, transplantation of a cell or cells into unusual conditions is typically employed.

The simplest experiment of this kind was performed on amphibian embryos. As mentioned earlier, it is possible to construct a fate map of the blastula or gastrula, containing detailed indications of which Organs of the adult organism will develop from specific regions of the embryo. It is easy to observe that under normal development, cells of one region give rise to the epidermis, while cells of another give rise to the Brain. To answer our question, it is necessary to swap two pieces of embryonic tissue excised from different regions—for example, so that a portion of the future (presumptive) epidermis ends up in THE PLACE OF the future brain, and vice versa. If the cells are already determined at the time of transplantation, they will develop autonomously in accordance with their previous position; i.e., cells from the presumptive epidermis region, when transplanted into the region destined to form the brain, will form epidermis, whereas cells from the future brain region, after transplantation into the region giving rise to the epidermis, will form neural tissue. Note, however, that at the early gastrula stage, cells still do not "remember" their origin and differentiate according to their new position. But if the same experiment is performed somewhat later (e.g., at the late gastrula stage), presumptive brain cells in the epidermal region will differentiate into neural tissue, and presumptive epidermal cells transplanted to the future brain region will differentiate into epidermis. Consequently, both groups of cells have become determined at some point between the early and late gastrula stages.

16.2.10. The state of determination can be determined by cytoplasm or chromosomes [24, 25]

Cellular memory is one of the most intriguing problems in molecular biology: what governs the maintenance of a specific pattern of gene expression? A detailed Discussion of this issue from a biochemical perspective can be found in Ch. 10. Here, for the purpose of further discussion, it is useful to divide molecular mechanisms into two classes, which can be designated as cytoplasmic and nuclear.

When we speak of cytoplasmic memory, we imply that components encoded by a specific set of genes are present in the cytoplasm (or in the extracellular environment) and exert a direct or indirect feedback action on the genome, maintaining the selective expression of a particular set of genes; the cytoplasm of different cell types contains distinct controlling factors. Thus, if a nucleus is obtained from differentiated cells of one type and injected into the cytoplasm of cells of another type, the pattern of gene expression should change to match the host cytoplasm. Nuclear transplantation experiments on amphibian eggs (see sect. 16.2.1), as well as other artificial Cell Fusion experiments, have provided evidence that this is precisely how it happens. This highlights the important role of mechanisms ensuring cytoplasmic memory.

At the same time, nuclear memory, also known as Genomic Imprinting, is determined by self-sustaining changes resulting from alterations in chromosome properties. During these changes, The nucleotide sequence of the DNA remains constant, but a selection is made as to which genes will be expressed. The best-studied examples of nuclear memory are associated with DNA Methylation: as explained in sect. 10.3.16, the existing pattern of cytosine methylation in DNA can be inherited across cell generations, a property maintained by the action of the methylase enzyme.

Embryonic development is akin to a natural experiment set up to study nuclear memory. The sperm and the unfertilized egg differ significantly in their state of cellular differentiation, yet they contain a virtually identical set of genes, and upon fertilization, their chromosomes combine within a single cell. Do the Functional differences between chromosomes originating from the sperm and the egg persist after their fusion in the zygote? Studies in mice have yielded a positive answer to this question.

16.2.11. Chromosome sets originating from the sperm and egg carry the imprint of their history [25]

As already mentioned, an unfertilized egg can be stimulated to undergo cleavage without the aid of sperm: whether activation occurs spontaneously or is artificially induced, the result is a parthenogenetic embryo (see sect. 16.2.6). In certain animal species, including vertebrates (e.g., some lizards), such an embryo is capable of developing into a normal, healthy adult animal. In mammals, parthenogenetic embryos die at early stages of development; in these animals, development without fertilization remains unknown despite great interest in the problem and numerous attempts by experimenters. The reason for these failures was uncovered in experiments on mouse eggs.

It is known that a fertilized egg (zygote) contains two pronuclei, one derived from the father and the second from the mother. Using a micropipette, one can extract one of the pronuclei and replace it with a pronucleus from another egg (Fig. 16-28). In this way, we create a zygote containing either two maternal or two paternal pronuclei. In either case, the genome will be the same (assuming the paternal pronucleus contained an X chromosome rather than a Y chromosome). It turned out that a zygote with two paternal pronuclei forms an embryo lacking the structures that normally develop from the inner cell mass, whereas a zygote with two maternal pronuclei forms an embryo lacking structures derived from the trophectoderm. Since both types of zygotes possess identical cytoplasm, these experiments strongly indicate that nuclear memory dictates the expression of different sets of genes on the maternal and paternal chromosomes; in other words, certain genes are expressed only when inherited from the father, while others are expressed when inherited from the mother.

Fig. 16-28. The pronuclear transplantation method makes it possible to obtain eggs in which both pronuclei belong to the male (or female). Membrane fusion occurs as a result of Treatment with Sendai virus inactivated to a certain degree, injected at the Third Stage simultaneously with pronuclear transplantation.

Observations on Transgenic Mice have shown that DNA methylation underlies The phenomenon of genomic imprinting. For instance, one can cross transgenic mice carrying foreign DNA sequences and obtain offspring that inherit the integrated foreign sequence exclusively from the father or exclusively from the mother. In such mice, the integrated sequences can be methylated differently depending on whether they originated from the sperm or the egg. This methylation pattern is maintained in somatic tissues throughout the adult animal's life, but it can change during the formation of germ-line cells for the next generation of Gametes. The expression pattern of one gene known to behave in this manner is determined by methylation; if the gene is inherited from the mother, it is methylated and not expressed in the given animal, whereas the same gene inherited from the father is unmethylated and expressed.

Conclusion

During development, a multitude of cells of various types arise from a fertilized egg. With rare exceptions, the genomes of differentiated cells remain unchanged; only the pattern of gene expression is altered. The differences arising between cells can be the consequence of an unequal distribution of cytoplasmic determinants in the egg prior to the onset of division, or the result of sequential Changes in the cellular environment within the embryo. For example, in Xenopus, blastomeres of the animal and vegetal hemispheres inherit different cytoplasmic determinants. Vegetal hemisphere blastomeres induce animal blastomeres to develop along the mesodermal pathway; in the absence of such an induction, animal blastomeres give rise to ectoderm. This induction is mediated by signaling molecules (FGF and TGF-β2 or their analogs), which participate in the Regulation of Cell growth and differentiation in adult organisms.

Mammalian eggs possess unique properties, being essentially symmetrical. Due to this property, all mammalian blastomeres are initially indistinguishable from one another; differences arise as a consequence of cell-cell interactions. For the same reason, cells from two different embryos combined into a single entity are able to adjust their developmental program and form a chimeric mouse. In the absence of normal influences from neighboring cells, blastomeres of an early mouse embryo can develop abnormally, forming teratocarcinomas, which are used to derive embryonic stem cells. Upon implantation into normal early embryos, such cells restore normal behavior; their descendants differentiate according to their environment and are capable of participating in the formation of a normal embryo.

During embryonic development, embryonic cells acquire specific differences that they must retain even after the disappearance of the stimulus that caused such cellular diversification. This requires cells to possess a memory that ensures cell determination toward a specific specialization long before overt signs of differentiation appear. Mechanisms of cellular memory can be either cytoplasmic—defined as the action of cytoplasmic molecules on the nucleus to maintain their own synthesis—or nuclear, based on Chromatin modifications such as DNA methylation.