Cell determination and differentiation

From the time a fertilized ovum begins to divide (cleavage) until it forms a complete organism, it passes through successive stages in which groups of cells become increasingly specialized. This process of cell specialization involves two key steps: determination, in which cells become committed to a certain developmental pathway, and differentiation, in which cells acquire their ultimate structure and function. The term differentiation may also be used in a broader sense to describe the entire process of cell specialization. A third process, called morphogenesis, is needed to mold the embryonic cells structurally into the various tissues and organ systems of the mature organism. For example, in eye development, the specialized photoreceptor cells of the retina are a product of determination and differentiation, but their organization into the overall structure of the eye is a product of morphogenesis.

The Process of Specialization

In animal development, the fertilized ovum (zygote) has the potential to develop into any type of cell in the body; it is said to be totipotent. As it undergoes cell division beyond the first few cleavages, the resulting cells (blastomeres) lose their totipotency. This point is reached at different stages in different species. In the sea urchin, for example, isolated cells of the two- or four-cell embryo can develop into completely new adults; in other species, such as the tooth shell clam (Dentalium), these early cleavage cells, when isolated, do not have the potential to develop into a complete organism. In all animals, the cells of later embryos lose their ability to form new organisms. This loss of developmental potency of cells is called restriction and is dictated to some extent both by the type of cytoplasm with which the cell is endowed and the influence of surrounding cells. As development progresses, restricted cells become “committed” to develop into a specific tissue. Under normal conditions, these cells will always develop into this designated tissue; if, however, the cells are moved experimentally to some other part of the embryo or to another embryo, they will develop in a different way. That is why commitment is said to be reversible, but determination is not.

Determination occurs when a group of cells becomes irreversibly assigned to develop into a specific tissue. Determination is the final step in restriction; beyond this point, the cells have no other developmental options. Determined cells may or may not look different from other embryonic cells, but they have changed internally so that they are committed to a particular developmental pathway. Determined cells are said to be self-perpetuating because they can pass on heritable information about their identity and do not require stimulation by surrounding cells to develop in a certain way.

Once the fate of a cell is determined, then differentiation (also called cell differentiation or cytodifferentiation) can take place. During differentiation, the cell undergoes structural and functional changes, which result in a highly specialized, mature, differentiated cell. For example, red blood cells become specialized by losingtheir nucleus and other organelles to fill themselves completely with the oxygen-carrying hemoglobin molecules. It also maximizes its surface area by becoming flattened and doughnut-shaped. Primitive muscle cells, called myoblasts, become specialized by fusing together to form elongated multinucleated cells called myotubes. These cells further specialize by forming contractile organelles called myofibrils, composed primarily of the proteins actin and myosin.

The Role of Genes

At one time, it was believed that the genetic determinants (genes) were divided up and parceled out as determination and differentiation progressed, such that each cell type received certain genes. This hypothesis was disproved in several ways, one of them being nuclear transplantation experiments, in which adult cell nuclei were transplanted into fertilized eggs whose nuclei had been removed or inactivated. A small percentage of these eggs were able to develop into normal adults, thus proving that the adult nuclei implanted in them retained all the genes necessary to form a complete organism. These and other experiments have led to the conclusion that both determination and differentiation occur because certain genes are expressed at certain times during the life history of the cell. There remains, however, the question of how genes are turned on and off to control development.

All developmental processes are believed to be controlled by genes as part of an intricate developmental program. The genes of individual cells are activated or deactivated via various signaling mechanisms to provide the correct cellular responses at the appropriate times. This process begins very early in development and even occurs in the egg before fertilization. Messenger ribonucleic acid (RNA) and proteins are produced by the egg and distributed unequally in the cytoplasm of different parts of the egg. When cleavage occurs, blastomeres in one part of the embryo receive cytoplasm that differs from the cytoplasm received by blastomeres in another part of the embryo; thus, some cells are endowed with one kind of messenger RNA and protein, and other cells with another kind. In some species, cleavage is even unequal to ensure that certain cells receive the desired cytoplasm. For example, in the nematode Caenorhabditis elegans, unequal cleavage results in the establishment of five different tissue types by the sixteen-cell stage. As the messenger RNA is expressed in the form of new proteins, it gives unique qualities to the cells. Some of these new proteins and the proteins made earlier in the egg may be signal molecules, which stimulate new and unique gene expression in the nucleus.

Another mechanism for turning genes on and off is called embryonic induction, which occurs when one embryonic tissue influences the development of another by releasing chemical factors called inductors. The inductors are signal molecules that instruct cells of another tissue how to develop by directly or indirectly activating certain genes. Induction is especially noticeable after the formation of distinct tissues, such as the three germ layers, including the ectoderm, endoderm, and mesoderm. In vertebrates, the mesoderm induces the formation of the neural tube and various other parts of the nervous system.

Another set of mechanisms, similar to embryonic induction, that controls differentiation involves the microenvironment in which the embryonic cells exist. These mechanisms include such parameters as the position of a cell in relation to other cells in the embryo; the interactions of cells with the extracellular milieu, including ions, pH, oxygen, and extracellular matrix proteins such as collagen; direct cell-to-cell contact; and the presence of specific growth and differentiation factors. The cells in one part of the developing embryo will experience a completely different set of microenvironmental influences from that of cells in another part of the embryo and, consequently, will be prompted to express their genes in different ways. Once the genes have been expressed, there must be a means for the daughter cells to retain the unique gene expression of the parent cell. That is most likely accomplished by proteins passed on to the daughter cells that continue to activate or deactivate the appropriate genes.

Genetic Recombination and Transcription

Although the genome is not modified extensively during embryonic development, there is evidence that certain changes occur to enhance differentiation. One of these mechanisms is called genetic recombination and involves breaking and rejoining deoxyribonucleic acid (DNA) at defined sites. For example, in maize, segments of DNA called transposons move around the genome and presumably take control of specific genes at certain times during development. Another example of genome modification occurs in the nematode Ascaris, in which parts of the chromosomes are discarded (in a process called chromosome diminution) during cleavage. The discarded chromatin is believed to be composed of extra copies of DNA sequences that are not ordinarily transcribed. The genome is also modified by making extra copies of essential genes (gene amplification). For example, some genes in the follicle cells surrounding the maturing oocyte of Drosophila are amplified about thirty times to code for the large amount of protein needed to make the egg chorion. As cells differentiate, the types of messenger RNA and protein they produce become increasingly selective so that, eventually, each cell type has a unique pattern of gene expression. There will always, however, be common genes expressed in every cell that is needed for basic housekeeping processes, such as respiration and transport.

Selective gene transcription is carefully controlled by various mechanisms involving the blocking and unblocking of DNA. Two classes of nuclear proteins are believed to be involved in switching genes on and off: histones and nonhistones. Histones associate with DNA molecules in such a way that they block the DNA from being transcribed. The nonhistones are believed to remove or rearrange histones so that the DNA can be replicated or transcribed. Some nonhistone proteins are gene-regulatory proteins which recognize a particular DNA sequence. The binding of these proteins with DNA can either facilitate or inhibit transcription. An additional control found only in vertebrates is methylation, by which methyl groups are added to the DNA base cytosine. In general, the inactive genes of vertebrates are more highly methylated than active genes; thus, methylation may serve to strengthen decisions involving gene expression that are made during differentiation.

Once the appropriate messenger RNA has been produced, it must still be translated into protein and the protein must be assembled and made functional. For example, hemoglobin protein (globin) translation is controlled by the presence of heme, the iron-containing portion of the hemoglobin molecule. In the absence of heme, the factor that initiates globin translation is inactivated; thus, even though the appropriate messenger RNA and other necessary ingredients are present, without heme no hemoglobin protein will be produced. Even after proteins are translated, they are subject to further regulatory mechanisms, such as assembly into functional units, activation or inactivation by various enzymes and other factors, and transport to their cellular destination.

Studying Embryology

The study of embryology was transformed from a purely descriptive science into an experimental science by investigators who developed microsurgical techniques in the 1890s. They discovered that the separated blastomeres of some early embryos, such as the sea urchin, could develop into complete normal larvae (regulative or indeterminate development) and that the blastomeres of others, such as the tunicate, could form only parts of embryos (mosaic or determinate development). This led to an appreciation of the importance of nuclear-cytoplasmic interactions and the fact that egg cytoplasm distribution plays an important role in determining how certain blastomeres develop. Further microsurgical separation studies on embryos in later stages (thirty-two-cell to sixty-four-cell stages) demonstrated that the microenvironment of some embryos approximates a double gradient consisting of animal pole factors in one half and vegetal pole factors in the other half. These two chemical gradients influence the cell nuclei in their respective zones and cause the cells to become progressively determined in certain ways. Thus, simply by manipulating embryonic cells, scientists were able to demonstrate the concepts of restriction and determination.

Another microsurgical technique developed in the first half of the twentieth century involves transplanting tissue from one embryo to another. When tissue that normally forms the brain is transplanted to an area of another embryo that normally forms skin, the transplanted tissue develops independently and begins to form a brain. Thus, the tissue has become irreversibly committed or determined to form a particular adult tissue. If the same transplant is done at an earlier stage, the transplanted tissue conforms to its surroundings and forms an epidermis. These results indicate that tissues are capable of changing their normal fate if they are influenced by another tissue before determination. In some instances, the transplanted tissue induces the surrounding tissue to change its normal fate. Such is the case when tissue from the dorsal lip of the blastopore of an amphibian gastrula is transplanted to the lateral lip area of another gastrula. The transplanted tissue induces the formation of a second complete embryonic axis, resulting in laterally conjoined twins.

Another elementary method that has yielded a large amount of information about determination is cell marking and tracing. At first, investigators took advantage of different natural pigments that are present in certain animal embryos by following the fate of each blastomere. They discovered that each colored cytoplasm gives rise to a specific embryonic fate. For example, the yellow crescent cytoplasm of the tunicate (Styela partita) embryo gives rise to adult muscle cells. In other studies, cells were marked with vital dyes, carbon particles, enzymes, radioactive labels, and distinctive cells transplanted from another embryo. By tracing these labeled cells, investigators were able to ascertain their ultimate fate and when and where cell determination takes place.

Embryologists needed to determine whether the nuclei of determined and differentiated cells are irreversibly modified. All the genetic material present in differentiated cells could be shown by microscopic observations of the chromosomes, especially the large polytene chromosomes of larval flies, such as Drosophila. The only way to show whether these chromosomes were functional, however, was to transplant the nucleus of a differentiated cell into an enucleated egg and see if it could direct the development of a complete organism. The technique of nuclear transplantation (sometimes called cloning), developed in the 1950s, did indeed prove that nuclei from differentiated cells are totipotent. Success was not universal with all tissue types, however, and only a small percentage of the transplants actually succeeded, which indicates that restriction of potency does involve changes in the nucleus but not permanent modification of the genome itself.

The study of embryology in the twenty-first century continues to uncover important processes and applications for understanding cell determination and differentiation. For example, in one study, scientists investigated the gene transcriptional stochasticity of several single-cell organisms at various stages of cell differentiation using transcriptional uncertainty. They found that in the early stages of cell differentiation, gene transcription bursts increase until the cell reaches the peak of decision-making and takes on a terminal identity. Advances in modern technology like Raman spectroscopy facilitate studies like these. Additionally, as scientists gain further knowledge of cell differentiation and determination processes and similar mechanisms, gene editing technology and assistive reproduction technology improve.

Methodology

The study of differentiation can be approached by several methods. The simplest is to observe tissues microscopically as they differentiate. That is done most often by fixing embryos at various stages of development and observing thinly sliced sections of them with a light or electron microscope. Another method is to explant cells, tissue, or organs from embryos and observe them in culture (in vitro). By doing so, scientists can manipulate the environment of the cultured cells to find out precisely what conditions are necessary for differentiation to occur. In vitro culture also facilitates techniques like synchronizing cell growth to study the relationship between cell division and cell differentiation and cell fusion to see how the contents of one cell affect the behavior of another.

Various biochemical and molecular techniques aid the study of the roles of many biological molecules in differentiation. Of particular interest are separation methods that allow scientists to isolate and identify proteins and other factors involved in the differentiation process. These molecules can be isolated by first homogenizing the cells and then separating the desired molecules by centrifugation (based on density), electrophoresis (based on electrical charge), or chromatography (based on molecular size). Once the molecules are isolated, their properties can be studied, including their biological activity. At times, it is important to know if a particular sequence of DNA or RNA is present in an embryonic cell. That can be determined by a technique called hybridization, whereby single-stranded DNA is allowed to match up and adhere to a complementary strand of DNA or RNA. Usually, one of the strands is radioactively labeled so that the sequence in question can be detected and measured. The usefulness of this technique has become greatly enhanced by the development of recombinant DNA technology, which allows for the construction of specific molecular probes (DNA sequences) that can be radiolabeled and used to detect cellular DNA and RNA by hybridization. Recombinant DNA technology has produced breakthrough medicines to treat patients with hemophilia A, breast cancer, stroke, kidney failure, and more. One step further is the technique called DNA transformation, whereby isolated genes are modified and then reintroduced into cells to determine the altered gene's new properties when expressed. DNA transformation technology is important for gene expression studies, genetic engineering, synthetic biology, and more.

Determination and differentiation are the foundations of cell diversification. There are hundreds of different eukaryotic cell types in humans and thousands more in all mammals. Without cell specialization, organisms would not be able to move, breathe, think, or perform any of the many other functions necessary to sustain life.

Principal Terms

Commitment: The “decision” by an embryonic cell to develop in a certain way, which may be reversed if the cell is removed from its normal surroundings

Embryonic Induction: The point at which one embryonic tissue signals another embryonic tissue to develop in a certain way

Genome: All the genes of one organism or species

Morphogenesis: The development of form, including the overall form of the organism and the form of each organ and tissue

Mosaic Development: The process whereby early embryonic cells are determined by the cytoplasm they receive from the egg; also called determinate development

Regulative Development: The process whereby early embryonic cells are determined by their interactions with other cells; also called indeterminate development

Restriction: Reduction of the developmental potency of a cell

Totipotent: The ability of a cell to develop into any kind of cell in the body

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