Chapter 2 Cell Potency and Fate 2024 PDF

CHAPTER 2 CHAPTER 2: C ELL POTENCY AND FATE Key terms and concepts: Genome, gene expression Chromosome, chromatin Cis-acting sequences, promoter, enhancer Transcription, translation mRNA intron, exon In situ hybridization (ISH), immunohistochemistry (IHC), PCR, west...

CHAPTER 2 CHAPTER 2: C ELL POTENCY AND FATE Key terms and concepts: Genome, gene expression Chromosome, chromatin Cis-acting sequences, promoter, enhancer Transcription, translation mRNA intron, exon In situ hybridization (ISH), immunohistochemistry (IHC), PCR, western blotting, Enzyme-linked immunosorbent assays (ELISA) Epigenetic modifications (DNA methylation, histone acetylation, histone methylation) DNA mutation Induction Cell potency: totipotent, pluripotent, multipotent, determined Stem cell Learning objectives: By the end of this unit, you should be able to: 1. Define the key terms and concepts listed above and understand how each relates to developmental biology. 2. Explain the difference between a genetic mutation versus an epigenetic modification of DNA. 3. Explain the impact of epigenetic DNA modification of gene expression and cellular differentiation. What are two potential mechanisms of epigenetic DNA modification? 4. Explain the role of transcription factors in the process of gene expression. 5. Understand the differences in cell fate between a totipotent cell and a differentiated cell and explain the role of gene and protein expression in determining those differences. 6. Be able to define the terms “totipotent”, “pluripotent”, “multipotent” and “determined” with respect to cells and describe the types of progeny that cells of each of the 4 potencies could form. 7. Understand how In situ hybridization, PCR, immunohistochemistry, and western blotting are used to assess gene and protein expression I. Concepts of Development A. The formation of a fetus from a single-cell requires a multitude of events that are precisely timed and integrated. The mechanisms controlling development are often only partially understood, but partial knowledge is still helpful. It is often errors in these mechanisms that cause abnormal development. B. The genome is the complete set of DNA sequence of an organism. Although for all intents and purposes, every adult cell contains the same genetic information as the original fertilized egg, individual types of mature cells express different genes. It is these different patterns of gene expression that determine the characteristics of each cell. The different proteins expressed result in observable functional and/or structural differences between cells. C. One of the primary goals of development is to produce a diverse array of cell types that are highly organized and form a body. One of the key questions in 1 C ELL POTENCY AND FATE developmental biology is how cells form the correct cell types in all the correct places at the correct time in the developing embryo. A great deal of scientific effort is expended trying to understand the regulation of gene expression in the embryo and how this gene expression controls the processes of embryogenesis. D. A brief review of transcription and translation (part of the “central dogma” of Molecular biology) 1. The double stranded DNA within the cell nucleus encodes the genetic information of the organism. All cells of the organism, with the unusual exception of lymphocytes, have the same DNA. 2. The nuclear DNA is packaged into chromatin (DNA/protein complex) by association with Histone proteins to form tightly packed nucleosomes (repeating units of chromatin). The tightly packed chromatin comprises the chromosomes. Each chromosome is composed of double stranded DNA packed into chromatin through its association with histone proteins to form nucleosomes. 3. The degree to which the chromatin is compacted can influence the expression of genes on the DNA strand. Tightly packed chromatin tends to be repressed. The packing of the chromatin can be regulated, for example by histone modifications such as acetylation or methylation, and is one way a cell can modulate gene expression. Acetylation tends to loosen chromatin and promote increased gene transcription. Chromatin methylation on the other hand tends to cause decreased gene transcription. 4. Genes within the DNA encode proteins that can be produced by the cell. In order for protein synthesis to occur, the DNA must first be TRANSCRIBED into RNA. The nuclear enzyme RNA polymerase synthesizes an RNA copy of the DNA strand. This process is called TRANSCRIPTION. 5. Transcription is a highly regulated process in cells. RNA polymerase must bind to the promoter region of a gene in order to initiate transcription. 6. Cis-acting DNA sequences are regulatory elements that reside on the same strand of DNA as the gene they regulate. Both promoter and enhancer regions are examples. An enhancer region is a DNA sequence that transcription factors bind to, altering the efficiency of and rate of transcription from a specific promoter. These regions can be very distant from the gene they regulate. 7. Transcription factors are proteins that activate or repress gene transcription. Transcription factor binding to cis- DNA sequences is a very important way cells regulate which genes are transcribed. 8. Transcription of the DNA by RNA polymerase results in the production of long nuclear RNA strands that consists of the templates for protein synthesis (the EXons, which will be EXpressed), as well as intervening sequences called INtrons (which are IN-between). 9. Some genes can be spliced in several different ways to produce proteins with different activities. This process is called Alternative Splicing and is an important mechanism of regulation of protein expression. 10. Once RNA processing is completed in the nucleus, the newly formed messenger RNA (mRNA) can be exported from the nucleus to the cytoplasm. 2  CHAPTER 2 11. Once in the cytoplasm, the mRNA can be TRANSLATED into protein by ribosomes. Translation is also a highly regulated process that can be modulated by cells to control the levels of protein expressed by a cell. 12. Once the protein has been synthesized by the ribosomes, it can be further regulated by processes such as protein degradation, post translational modification to even further fine-tune the cell’s control over protein activity. Transcription/translation schematic: 3 C ELL POTENCY AND FATE Several techniques are commonly applied for detecting both the amount and localization of mRNA and protein in tissues and cells. The quantity of mRNA, which is a measure of gene expression is often assayed by polymerase chain reaction (PCR), following reverse transcription of mRNA into cDNA. In situ hybridization (ISH) uses a labeled complementary RNA strands to localize the cellular and tissue expression of specific mRNA transcripts. A technique called western blotting is used to determine relative quantities of specific proteins in cells or tissues while immunohistochemistry (IHC) is a technique commonly used to assess the cellular and tissue localization of protein. Both of these techniques take advantage of the principle of antibody binding specificity. Enzyme-linked immunosorbent assays (ELISA) also apply the principle of antibody binding specificity to detect proteins (including peptides and antibodies) and hormones. Molecular biology techniques to detect mRNA, DNA, and protein are widely used in research and in clinical applications. Appropriate application of these techniques and interpretation of assay results is dependent upon understanding their molecular basis. E. The differences that you will observe in histology among cell types are ultimately due to several factors. 1. The physical characteristics and biochemical activities of cells are determined by the patterns of protein expression in the cells. Protein expression can be regulated at the level of which genes are transcribed into messenger RNA (transcriptional regulation), RNA processing including splicing, which mRNA’s are translated into protein (translational regulation), as well as which mRNA’s are degraded (regulation of mRNA stability), protein stability, and other post- translational means of modulating protein, such as phosphorylation, specific cleavage, or localization within the cell. 2. The genome is the complete set of DNA sequence of an organism. Although for all intents and purposes, every adult cell contains the same genetic information as the original fertilized egg (with the unusual exception of lymphocytes), different parts of the genome will be "turned on" in some cells and not in others. 3. Epigenetic modification of DNA; another mechanism of modulation of gene expression is modification of the DNA in cells to impact its transcription. The basic genetic information (the nucleotide bases) of the DNA remains the same. However, methyl groups added to DNA (DNA methylation) or modifications to the histone proteins that compact the DNA into chromatin (for example acetylation or methylation of histones) can alter the accessibility of the DNA to the transcriptional machinery and (semi)permanently modify the ability of the affected segments of DNA to be transcribed. These modifications are termed “epigenetic” DNA modifications”, and can be heritable from mother cells to daughter cells. As such, these changes can exert transgenerational effects. 4. DNA mutation- mutations in DNA are permanent changes to the base-pair sequence of the DNA in the nucleus. These changes are permanent, are passed on to the progeny of the affected cell, and can have very profound effects on the expression of a gene and/or the activity of the protein the gene encodes. 5. Induction - During development, cells are often influenced by close range communication. One cell population may instruct another to take on certain characteristics. This process is called induction. Induction is defined as the process by which one group of cells influences another at close range. 4  CHAPTER 2 II. Cell Fate Commitment A. Cell fate: Each organism begins as a single cell that divides repeatedly to produce an adult organism with perhaps billions of cells! As soon as there are differences in the local environment of cells or in interactions between and among cells, cells begin to be distinct from each other. These differences can be attributed to changes in patterns of gene expression and/or chromatin modifications. Changes in what each cell and its daughter cells can become are the result. What the cell and its daughters will become is referred to as their Cell fate. B. Commitment: The process by which a cell's fate becomes set is called commitment. Commitment can be subdivided into three stages: specification, determination, and differentiation. 1. Specification: The first stage of commitment of cell or tissue fate during which the cell or tissue is capable of differentiating autonomously (i.e. by itself) when placed in an environment that is neutral with respect to the developmental pathway. At the stage of specification, cell commitment is transient and still capable of being reversed. 2. Determination: a. The changes of determination involve the selection of a specific developmental path, not just maturation. Thus, one cell may be determined in the muscle lineage and will mature to create a muscle cell while another cell may be determined to the neural lineage and will mature into a neuron or other neural cell. Both cells undergo maturation, but along very different pathways. b. Determination appears to be the result of the turning on and off of genes. These changes are often not observable by standard means. Observable changes in the cell as a result of these chemical changes (differentiation) may not be seen for quite a while. c. A determined cell will usually eventually differentiate and begin to express proteins characteristic of its cell fate. These changes in protein expression will eventually produce the differences we can observe between cells. 5. Differentiation- The process by which an unspecialized cell becomes specialized into one of the many cell types that make up the body. The cell’s phenotype comprises its patterns of gene and protein expression, which generate cell morphology and physiologic activity. Differentiation may occur long after specification and determination. 6. Prior to commitment, cells of the early mammalian embryo are totipotent, and so could still become any cell of the body (they possess "total" potential). They then undergo a progressive restriction of their possible fates as development proceeds. 7. Cell potency: We use specific vocabulary to indicate how much potential different cells have: a. Totipotent- cells have “total” potency and can form any cell type of the body and most of the extra-embryonic membranes (fetal placenta). Blastomeres (chapter 4) are examples of totipotent cells. 5 C ELL POTENCY AND FATE b. Pluripotent- Cells have a “plurality” of potency and can form many cell types. This word is used to designate cells that can form any cell of the body but NOT all of the extra-embryonic membranes. c. Multipotent- cells have “multiple” potency and can form several different (but generally related) cell types of the body. An example is bone marrow stem cells that can form may different types of blood cells but not many other cells of the body. d. Determined- restricted to a single cell fate. III. Differentiation A. Differentiation can be defined as an overt display of different phenotypes by cells of the same genotype. In other words, it refers to observable or measurable specialization of cell structural or functional characteristics among cells that have the same genetic makeup. For example, the determination among cells as to which will become muscle or connective tissue is made long before it is expressed in overt differentiation (changes you can see), and is presumably recorded in the cells as a chemical distinction (turning on and off of genes) that is not grossly apparent. B. In general, the more differentiated a cell is, the less likely it will be to undergo mitosis. C. Some cells can remain determined, but undifferentiated, throughout life. We use the term somatic (or adult) stem cells to describe cells in the adult that are determined but undifferentiated. They are no longer totipotent, since you couldn't produce an entire embryo from them. For example, there are stem cells that can only produce new skeletal muscle cells. Unlike their highly differentiated relatives of the same cell type (i.e. skeletal muscle cells), they are capable of rapid mitosis when properly stimulated and can then differentiate into the mature adult form. Some stem cells are multipotent, and so are able to differentiate into more than one related cell type. Stem cells in bone marrow can become several types of blood cells, and so are multipotent. Stem cells are particularly important in providing the possibility for repair of highly differentiated tissues. D. Stem Cells- Definition: A stem cell is a relatively undifferentiated cell that can undergo cell division and give rise to 1. One progeny that is undifferentiated and remains a stem cell and 2. One progeny that goes on to differentiate and give rise to either one or several different mature cell types dependent upon whether the stem cell is totipotent, pluripotent, multipotent, or tissue specific (determined). 6