19 Differential Gene Expression in Development 19 Differential Gene Expression in Development 19.1 What Are the Processes of Development? 19.2 How Is Cell Fate Determined? 19.3 What Is the Role of Gene Expression in Development? 19.4 How Does Gene Expression Determine Pattern Formation? 19.5 Is Cell Differentiation Reversible? 19 Differential Gene Expression in Development Stem cells are actively dividing, unspecialized cells that have the potential to produce different cell types. In stem cell therapy, stem cells are injected into damaged tissues, where they will differentiate and form new, healthy tissues. Opening Question: What are other uses of stem cells derived from fat? 19.1 What Are the Processes of Development? Development: the process in which a multicellular organism undergoes a series of progressive changes that characterizes its life cycle. In its earliest stages, a plant or animal is called an embryo. The embryo can be protected in a seed, an egg shell, or a uterus. Figure 19.1 From Fertilized Egg to Adult (Part 1) Figure 19.1 From Fertilized Egg to Adult (Part 2) 19.1 What Are the Processes of Development? Four processes of development: • Determination sets the fate of the cell • Differentiation—the process by which different types of cells arise • Morphogenesis—organization and spatial distribution of differentiated cells • Growth—increase in body size by cell division and cell expansion 19.1 What Are the Processes of Development? Determination and differentiation occur largely because of differential gene expression. Cells in the early embryo arise from repeated mitoses and soon begin to differ in terms of which genes are expressed. 19.1 What Are the Processes of Development? Morphogenesis involves differential gene expression and the interplay of signals between cells. It occurs in several ways: • Cell division • Cell expansion in plants (position and shape are constrained by cell walls) 19.1 What Are the Processes of Development? • Cell movements are important in animals • Apoptosis (programmed cell death); essential in organ development Growth occurs by increasing the number of cells or enlargement of existing cells. 19.1 What Are the Processes of Development? Cell fate: which type of tissue the cell will ultimately become. Cell fate is usually determined quite early in development. The timing can be determined by transplanting cells from one embryo to a different region in a different embryo. Figure 19.2 A Cell’s Fate Is Determined in the Embryo 19.1 What Are the Processes of Development? Cell fate determination is influenced by gene expression and the extracellular environment. Determination is a commitment. Determination is followed by differentiation—the changes in biochemistry, structure, and function that result in different cell types. 19.1 What Are the Processes of Development? During animal development, cell fate becomes progressively more restricted. Cell potency: potential to differentiate into other cell types. • Totipotent—can differentiate into any cell type (early embryo) • Pluripotent—can develop into most cell types, but cannot form new embryos 19.1 What Are the Processes of Development? • Multipotent—can differentiate into several related cell types • Unipotent—can produce only one cell type: their own (mature organism) Many of these processes can be manipulated in the laboratory. 19.2 How Is Cell Fate Determined? How does one egg cell produce so many different cell types? Two processes for cell determination: • Cytoplasmic segregation (unequal cytokinesis) • Induction (cell-to-cell communication) 19.2 How Is Cell Fate Determined? Cytoplasmic segregation: • Factors within a zygote or egg are not distributed evenly and end up in different daughter cells after division. • Polarity—developing a “top” and a “bottom.” Can develop very early; yolk and other factors are distributed asymmetrically. 19.2 How Is Cell Fate Determined? In animal development, the animal pole is the top, the vegetal pole is the bottom. Sea urchin embryos 19.2 How Is Cell Fate Determined? If the sea urchin eight-cell embryo is cut vertically, it develops into two small larvae. If it is cut horizontally, the bottom develops into a larva, the top remains embryonic. This indicates that the top and bottom halves have already developed distinct fates. 19.2 How Is Cell Fate Determined? Model of cytoplasmic segregation: Cytoplasmic determinants are distributed unequally in the egg cytoplasm. Includes specific proteins, regulatory RNAs, and mRNAs that play a role in development of many organisms. Figure 19.3 The Principle of Cytoplasmic Segregation 19.2 How Is Cell Fate Determined? The cytoskeleton contributes to asymmetric distribution of cytoplasmic determinants: • Microtubules and microfilaments have polarity. • Cytoskeletal elements can bind motor proteins that transport the cytoplasmic determinants. 19.2 How Is Cell Fate Determined? In sea urchin eggs, a protein binds to the growing (+) end of a microfilament and to an mRNA encoding a cytoplasmic determinant. As the microfilament grows toward one end of the cell, it carries the mRNA along with it. The asymmetrical distribution of the mRNA leads to a similar distribution of the protein it encodes. 19.2 How Is Cell Fate Determined? Induction: cells in a developing embryo influence one another’s developmental fate via chemical signals and signal transduction mechanisms. 19.2 How Is Cell Fate Determined? Development of the lens in the vertebrate eye: The forebrain bulges out to form optic vesicles, which come in contact with cells at the surface of the head. These surface cells ultimately become the lens. The optic vesicle must contact the surface cells, or the lens will not develop. Figure 19.4 Embryonic Inducers in Vertebrate Eye Development 19.2 How Is Cell Fate Determined? The surface cells receive a signal, or inducer, from the optic vesicles. Inducers trigger sequences of gene expression in the responding cells. How genes are switched on and off to govern development is studied using model organisms. 19.2 How Is Cell Fate Determined? Vulval development in Caenorhabditis elegans: Adults are hermaphroditic; eggs are laid through a ventral pore called the vulva. Figure 19.5 Induction during Vulval Development in Caenorhabditis elegans (Part 1) 19.2 How Is Cell Fate Determined? During development, one cell, the anchor cell, induces the vulva to form from six cells on the ventral surface. Two signals are involved: the primary (1) and secondary (2) inducers. The concentration gradient of the primary inducer (LIN-3) is key. It is produced by the anchor cell and diffuses out to form the gradient. Figure 19.5 Induction during Vulval Development in Caenorhabditis elegans (Part 2) 19.2 How Is Cell Fate Determined? The inducers control activation or inactivation of genes through signal transduction cascades. This differential gene expression leads to cell differentiation. 19.3 What Is the Role of Gene Expression in Development? All cells in an organism have the same genes, but each cell expresses only certain ones. The mechanisms that control gene expression during cell fate determination and differentiation work at the level of transcription. 19.3 What Is the Role of Gene Expression in Development? Cell fate determination can occur by induction. When an inducer molecule binds to a receptor on the cell surface, a signal transduction pathway leads to activation of transcription factors. Figure 19.6 Induction 19.3 What Is the Role of Gene Expression in Development? Development is often controlled by these kinds of molecular switches, which allow a cell to proceed down one of two alternative paths. In nematodes, LIN-3 is a growth factor; it binds to receptors on vulva precursor cells, starting a signal transduction pathway that includes Ras protein and MAP kinases. 19.3 What Is the Role of Gene Expression in Development? The gene for b-globin (part of hemoglobin) is expressed in red blood cells. This gene exists in other cells, but is not expressed. This can be shown using nucleic acid hybridization. A probe for the b-globin gene will find its complement in DNA from brain cells but not in mRNA from brain cells. 19.3 What Is the Role of Gene Expression in Development? Differentiation in muscle cells: Muscle precursor cells come from an embryonic layer called the mesoderm. When these cells commit to becoming muscle cells, they stop dividing. In most embryonic cells, cell division and cell differentiation are mutually exclusive. 19.3 What Is the Role of Gene Expression in Development? • Cell signaling activates the gene for a transcription factor called MyoD. • MyoD activates the gene for p21, an inhibitor of cyclin-dependent kinases that normally stimulate the cell cycle. • The cell cycle stops so that differentiation can begin. Figure 19.7 Transcription and Differentiation in the Formation of Muscle Cells 19.4 How Does Gene Expression Determine Pattern Formation? Pattern formation: The process that results in the spatial organization of tissues and organisms. • Linked to morphogenesis, creation of body form Morphogenesis involves cell division and differentiation, as well as apoptosis (programmed cell death). 19.4 How Does Gene Expression Determine Pattern Formation? In human embryos, connective tissue links the fingers and toes. Later, the cells between the digits die. In-Text Art, Ch. 19, p. 399 19.4 How Does Gene Expression Determine Pattern Formation? Model organisms are used to study apoptosis. Mutants with altered cell death phenotypes are used to identify the genes and proteins involved. 19.4 How Does Gene Expression Determine Pattern Formation? C. elegans produces exactly 1,090 somatic cells as it develops, but 131 of those cells die. The sequential activation of two proteins controls this cell death. A third gene codes for an inhibitor of apoptosis in cells not programmed to die. Figure 19.8 Pathways for Apoptosis 19.4 How Does Gene Expression Determine Pattern Formation? A similar system controls apoptosis in human development. The proteins are similar to those of C. elegans. The conservation of this pathway in evolution indicates its importance: Mutations are harmful, and evolution selects against them. 19.4 How Does Gene Expression Determine Pattern Formation? Flowers are composed of four organ types (sepals, petals, stamens, carpels) arranged around a central axis in whorls. In Arabidopsis thaliana, flowers develop from a meristem (undifferentiated, rapidly growing cells) at the growing point on the stem. Figure 19.9 Organ Identity Genes in Arabidopsis Flowers (Part 1) 19.4 How Does Gene Expression Determine Pattern Formation? The identity of each whorl is determined by organ identity genes: • Class A genes, expressed in sepals and petals • Class B genes, expressed in petals and stamens • Class C genes, expressed in stamens and carpels 19.4 How Does Gene Expression Determine Pattern Formation? The genes code for transcription factors, which are active as dimers. Dimer composition determines which whorl will develop. The A, B, and C proteins, and many other plant transcription factors, have a DNA-binding domain called the MADS box. Figure 19.9 Organ Identity Genes in Arabidopsis Flowers (Part 2) 19.4 How Does Gene Expression Determine Pattern Formation? Two lines of experimental evidence support this model for floral organs: • Loss-of-function mutations— mutation in A results in no sepals or petals • Gain-of-function mutations— promoter for C can be coupled to A, resulting in only sepals and petals. (Homeotic mutation: one organ is replaced by another.) 19.4 How Does Gene Expression Determine Pattern Formation? A protein called LEAFY controls transcription of organ identity genes. Plants with loss-of-function mutations of LEAFY do not produce flowers. Transgenic orange trees, expressing the LEAFY gene coupled to a strongly expressed promoter, flower and fruit years earlier than normal trees. 19.4 How Does Gene Expression Determine Pattern Formation? Fate of a cell is often determined by where the cell is. Positional information often comes in the form of an inducer called a morphogen, which diffuses from one group of cells to another, setting up a concentration gradient. 19.4 How Does Gene Expression Determine Pattern Formation? A morphogen: • Directly affects target cells • Different concentrations of the morphogen cause different effects The “French flag model” explains morphogens and can be applied to differentiation of the vulva in C. elegans. Figure 19.10 The French Flag Model 19.4 How Does Gene Expression Determine Pattern Formation? Vertebrate limb development also follows the French flag model. Cells that develop into digits must receive positional information. Cells in the zone of polarizing activity (ZPA) secrete a morphogen called Sonic hedgehog (Shh). It forms a gradient that determines the posterior–anterior axis. Figure 19.11 Specification of the Vertebrate Limb and the French Flag Model 19.4 How Does Gene Expression Determine Pattern Formation? Morphogens have been studied in the fruit fly Drosophila melanogaster. The head, thorax, and abdomen are each made of several fused segments; different body parts arise from different segments (e.g., wings and antennae). Segments appear early in development, in the early larval stage. Cell fates have already been determined. 19.4 How Does Gene Expression Determine Pattern Formation? In the first 12 mitotic divisions there is no cytokinesis, forming a multinucleate embryo. Morphogens can diffuse easily in the embryo. In-Text Art, Ch. 19, p. 402 19.4 How Does Gene Expression Determine Pattern Formation? The steps of cell determination were studied using experimental genetics: • Developmental mutations were identified • Mutants were compared with wild types to identify genes and proteins • Experiments confirmed gene and protein functions 19.4 How Does Gene Expression Determine Pattern Formation? The experiments revealed a cascade of gene expression. Three gene classes are involved: • Maternal effect genes set up the major axes of the egg. • Segmentation genes determine boundaries and polarity of each segment. • Hox genes determine which organ will be made at a given location. 19.4 How Does Gene Expression Determine Pattern Formation? Maternal effect genes Transcribed in cells of the mother’s ovary; the mRNAs are passed to the egg. Bicoid and nanos help determine the anterior–posterior axis of the embryo. Figure 19.12 Concentrations of Bicoid and Nanos Proteins Determine the Anterior–Posterior Axis (Part 1) 19.4 How Does Gene Expression Determine Pattern Formation? mRNA from a third gene, hunchback, is distributed evenly in the embryo at first, but Nanos inhibits its translation, while Bicoid stimulates it—setting up a gradient of Hunchback. Figure 19.12 Concentrations of Bicoid and Nanos Proteins Determine the Anterior–Posterior Axis (Part 2) 19.4 How Does Gene Expression Determine Pattern Formation? How were these pathways determined? • Bicoid mutants produce larvae with no head and no thorax. Cytoplasm from the anterior end of wild-type eggs will produce normal larvae. • Cytoplasm from the anterior end of wild-type eggs, injected into posterior end of another egg, will produce anterior structures there. 19.4 How Does Gene Expression Determine Pattern Formation? • Nanos mutants produce larvae with no abdomen. Cytoplasm from the posterior end of wild-type eggs will produce normal larvae. 19.4 How Does Gene Expression Determine Pattern Formation? Segmentation genes Three classes of genes act in sequence: • Gap genes organize broad areas; mutations result in omission of several body segments. • Pair rule genes divide embryo into units of two segments each; mutations result in every other segment missing. 19.4 How Does Gene Expression Determine Pattern Formation? • Segment polarity genes determine boundaries and anterior–posterior organization in individual segments. Mutations result in posterior structures being replaced by reversed (mirror-image) anterior structures. Figure 19.13 A Gene Cascade Controls Pattern Formation in the Drosophila Embryo 19.4 How Does Gene Expression Determine Pattern Formation? Hox genes encode transcription factors that are expressed in different combinations along the length of the embryo. They determine cell fate in each segment. Hox genes are on chromosome 3 in the same order as the segments whose functions they determine. Figure 19.14 Hox Genes in Drosophila Determine Segment Identity 19.4 How Does Gene Expression Determine Pattern Formation? Hox genes are shared by all animals. They are homeotic genes—a mutation can result in one organ being replaced by another. 19.4 How Does Gene Expression Determine Pattern Formation? Clues to hox gene function came from homeotic mutants. Antennapedia mutation—legs grow in place of antennae. Bithorax mutation—an extra pair of wings grow. Figure 19.15 A Homeotic Mutation in Drosophila 19.4 How Does Gene Expression Determine Pattern Formation? Hox genes have a 180 base pair sequence called the homeobox. It encodes a 60 amino acid sequence called the homeodomain. The homeodomain binds to specific DNA sequences in the promoters of target genes. 19.4 How Does Gene Expression Determine Pattern Formation? This homeodomain is found in transcription factors that regulate development in many other animals with an anterior–posterior axis. 19.5 Is Cell Differentiation Reversible? A zygote is totipotent—it can give rise to every cell type in the organism. As development proceeds, cells become determined and lose their totipotency. But most differentiated cells still contain the entire genome and still have the capacity for totipotency. 19.5 Is Cell Differentiation Reversible? In 1958, experiments showed that an entire carrot plant could be cloned from differentiated carrot root cells. This showed that the root cell contained a functional, entire genome. In forestry and agriculture, many plants are produced as clones from a single cell, to produce uniform characteristics. Figure 19.16 Cloning a Plant 19.5 Is Cell Differentiation Reversible? In animals, early embryonic cells have totipotency. This permits genetic screening and some types of assisted reproductive technologies. An embryo can be isolated and one or a few cells removed and examined for certain genetic conditions. The remaining cells can develop into a complete embryo and be implanted into the mother’s uterus. 19.5 Is Cell Differentiation Reversible? An isolated animal embryo cell will not develop into a complete organism, but the nucleus has the potential to do this. Nuclear transfer experiments show that genetic material from a single cell can be used to clone animals. Frogs were cloned in the 1950s. 19.5 Is Cell Differentiation Reversible? These cloning experiments indicated that: • No genetic information is lost as the cell passes through developmental stages (genomic equivalence). • The cytoplasmic environment can modify the cell’s fate. 19.5 Is Cell Differentiation Reversible? In 1996, the first mammal was cloned by somatic cell nuclear transfer. A somatic cell from one sheep (the donor) was fused with an enucleated egg from another sheep. The donor’s cell was fully differentiated. After the fused cell began divisions, it was implanted into the uterus of a third sheep. Figure 19.17 Cloning a Mammal Working with Data 19.1: Cloning a Mammal Cloning of Dolly the sheep demonstrated that under appropriate circumstances, animal cells are totipotent. But there is a danger of premature aging. Dolly developed severe arthritis; premature aging may be due to shortened telomeres in her cells. Working with Data 19.1: Cloning a Mammal Question 1: In addition to mammary epithelium (ME) cells, Wilmut’s team also attempted cloning by nuclear transfer from fetal fibroblasts (FB) and embryo-derived cells (EC). What can you conclude about the efficiency of this cloning process? Working with Data 19.1, Table 1 Working with Data 19.1: Cloning a Mammal Question 2: Compare the efficiencies of cloning using nuclear donors from different sources. What can you conclude about the ability of different nuclei to be reprogrammed? Working with Data 19.1: Cloning a Mammal Question 3: Polymorphic DNA markers were used to analyze Dolly’s genetic make-up. The data for four short tandem repeat (STR) markers (FCB 11, FCB 304, MAF 33, and MAF 209) are shown in the figure. Working with Data 19.1: Cloning a Mammal In the electrophoresis gels, different genotypes produce DNA bands of different sizes. A sample of Dolly’s DNA was compared with samples from her nuclear donor (mammary cells from a Finn Dorset ewe) and from the recipient (her surrogate mother, a Scottish Blackface ewe). Working with Data 19.1, Figure A Working with Data 19.1: Cloning a Mammal Are the DNA bands from Dolly the same sizes as those from her nuclear donor or from her surrogate mother? What does this indicate about Dolly’s genetic makeup? 19.5 Is Cell Differentiation Reversible? Cloning of Dolly the sheep showed that a fully differentiated cell from a mature animal can revert to a totipotent state. Many other species have since been cloned by nuclear transfer. 19.5 Is Cell Differentiation Reversible? Reasons to clone animals: • Increase number of valuable animals, such as transgenic animals carrying genes with therapeutic properties. Example: a cow that was genetically engineered to make human growth hormone in milk has been cloned to produce the hormone for children with growth hormone deficiency. 19.5 Is Cell Differentiation Reversible? • Preservation of endangered species: Cloning may be the only way to save endangered species with low reproduction rates, such as pandas. • Preservation of pets 19.5 Is Cell Differentiation Reversible? Stem cells: rapidly dividing, undifferentiated cells that can differentiate into several cell types. In plants, stem cells are in the meristems In mammals, stem cells occur in tissues that require frequent replacement—skin, blood, intestinal lining. 19.5 Is Cell Differentiation Reversible? Stem cells in adult animals are multipotent: the daughter cells differentiate into only a few cell types. In the bone marrow, hematopoietic stem cells produce blood cells, mesenchymal stem cells produce bone and connective tissue cells. 19.5 Is Cell Differentiation Reversible? Multipotent stem cells differentiate “on demand.” Bone marrow stem cells differentiate in response to specific growth factors. This is the basis of a cancer therapy called hematopoietic stem cell transplantation (HSCP). 19.5 Is Cell Differentiation Reversible? Therapies that kill cancer cells can also kill other rapidly dividing cells such as bone marrow stem cells. The stem cells are removed, stored during the therapy, then returned to the bone marrow. The stored stem cells retain their ability to differentiate. Figure 19.18 Stem Cell Transplantation 19.5 Is Cell Differentiation Reversible? Adjacent cells can influence stem cell differentiation. Experiments show that damaged tissues can heal more effectively if stem cells are injected into the tissue. The mechanism is unclear; the stem cells may be able to insert into the tissue and differentiate, or signals from the stem cells induce tissue regeneration. 19.5 Is Cell Differentiation Reversible? In the embryonic stage called the blastocyst, a group of cells is pluripotent—they can differentiate into most cell types, but cannot give rise to a complete organism. In mice, these embryonic stem cells (ESCs) can be removed from the blastocyst and grown in laboratory culture almost indefinitely. 19.5 Is Cell Differentiation Reversible? ESCs can be induced to differentiate in the laboratory by specific signals. Treatment with a vitamin A derivative causes them to form neurons; other growth factors induce them to form blood cells. 19.5 Is Cell Differentiation Reversible? ESC cultures have potential as sources of differentiated cells to repair specific tissues, such as a damaged pancreas in diabetes or a brain that malfunctions in Parkinson’s disease. 19.5 Is Cell Differentiation Reversible? ESCs can be harvested from human embryos conceived by in vitro fertilization, with consent of the donors. However: • Some people object to the destruction of human embryos for this purpose. • The stem cells could provoke an immune response in a recipient. 19.5 Is Cell Differentiation Reversible? Another approach: induced pluripotent stem cells (iPS cells) can be made from skin cells. Genes essential to the undifferentiated state and function of stem cells were identified. These genes were coupled to highly expressing promoters and injected into skin cells. Figure 19.19 Two Ways to Obtain Pluripotent Stem Cells 19.5 Is Cell Differentiation Reversible? The skin cells were then pluripotent and could differentiate into many cell types. iPS cells can be made from a patient’s own skin cells, so immune responses are avoided. These therapies have been tested in animals. 17 Answer to Opening Question In the United States, veterinarians use multipotent stem cells derived from fat to treat injuries and osteoarthritis in animals. In the operating room, large quantities of stem cells can be isolated from human patients and used immediately to repair tissues, for example, after surgery for breast cancer.