BIOL 366: Mechanisms of Development Lecture Notes PDF

BIOL 366: Mechanisms of Development Lecture 1: Introduction to Developmental Biology Instructor: Amandeep Glory, PhD Office: SP375.23 Email: [email protected] Textbook: Developmental Biology, Scott F. Gilbert, 13th Edition (material will be predominantly based on the textbook,...

BIOL 366: Mechanisms of Development Lecture 1: Introduction to Developmental Biology Instructor: Amandeep Glory, PhD Office: SP375.23 Email: [email protected] Textbook: Developmental Biology, Scott F. Gilbert, 13th Edition (material will be predominantly based on the textbook, but I may use supplementary material from other sources) Teaching assistant: Alexandra Perlman Email: [email protected] Office: SP301.17 Office hours: by appointment Marking scheme Assessment Date % Online quizzes see quiz schedule 10 Midterm 1 11-Oct 20 Midterm 2 15-Nov 20 Final Exam (cumulative) according to exam office 50 Alternative marking scheme *if your final exam grade is better than one of the two midterms Assessment Date % of final grade Online quizzes (4) See quiz schedule 10% (lowest quiz grade dropped) Midterms (2) Midterm 1: Friday, October 6th Lower midterm (covering material from lectures 1-8) grade – 10% Midterm 2: Friday November 15th Higher midterm (covering material from 9-16) grade – 20% Final exam TBD according to final exam office 60% (cumulative) *This alternative grading scheme is only available if you have written both midterms. Alternative marking scheme *if you miss one of the midterms Assessment Date % of final grade Online quizzes (4) See quiz schedule 10% (lowest quiz grade dropped) Midterms (2) Midterm 1: Friday, October 6th 20% (covering material from lectures 1-8) Midterm 2: Friday November 15th (covering material from 9-16) Final exam TBD according to final exam office 70% (cumulative) Quiz schedule Quiz opens at 10:00 Quiz closes at 22:00 Lectures covered Quiz # 16-Sep 20-Sep 1, 2, 3, 4 1 30-Sep 04-Oct 5, 6, 7, 8 2 28-Oct 01-Nov 9, 10, 11, 12 3 11-Nov 14-Nov 13, 14, 15, 16 4 25-Nov 29-Nov 17, 18, 19, 20 5 The quiz with the lowest grade will be dropped (best 4 out of 5 quizzes) Course description Survey course covering the basic principles and molecular mechanisms of animal development. Course topics Introduction to developmental biology Specifying cell identity Differential gene expression Cell-cell communication Stem cells Gametogenesis Fertilization Specific examples of animal development: Snail, C. elegans, Drosophila, sea urchin, amphibians, birds, mammals Early human development Textbook Chapter 1 Pages 1 – 8, 14 – 20 Chapter overview Introduction to development Model organisms Fate maps + embryo observation What is development? Multicellular organisms are generated Example of planarian regeneration: from single cells through a slow progressive change called development. Organisms never stop developing. Tissues can regenerate continuously (e.g. skin cells, red blood cells) Tissues can regenerate after injury (e.g. liver cells, planarians) Some organisms go through metamorphosis (e.g. caterpillars à butterflies) https://www.med.hku.hk/en/news/press/20210426-collagen-iv- planarian-stem-cell What is development? Development accomplishes two major objectives: 1. Generating cellular diversity and order within the individual organism (i.e. creating different organs and tissues from a single cell and making sure they are in the right place in the adult body) 2. Ensuring the continuity of life from one generation to the next  Developmental biology seeks to answer these questions about development: Differentiation: how can identical sets of genetic instructions produce different types of cells? Genetically identical cells Example of C. elegans embryo development: are destined to become different cell types. Draper et al. 1996 Cell  Pattern formation: What processes control tissue and organ patterning in the body? How does the body know that the liver is supposed to be on the right side of the body? Morphogenesis: What processes control tissue and organ shape? How does the body know what the liver is supposed to look like?  Growth: How do our cells know when to stop dividing? How is cell division so tightly regulated? Reproduction: How do germ cells form the next generation? Human development: How do we develop and how do outside factors interfere with human development? Environmental integration: How do environmental cues affect development? Example: If turtle eggs incubate below 27.7ºC, the hatchlings will be male. If they incubate above 31ºC they will be female. Evolution: How do changes in development create new body forms?  Regeneration: How do stem cells remain undifferentiated and how do they replenish themselves? Regenerative medicine and stem cell therapy: Many clinical trials are in progress to use stem cells to treat diseases such as Alzheimer’s, Parkinson’s, spinal cord injuries, heart failure, diabetes, and macular degeneration (among many others). However, while it remains a promising area of research, scientists and doctors need to remain cautious as unapproved therapies have caused more harm than good. We need to learn more about how stem cells migrate in the body and how they differentiate. How do we answer these questions? Model organisms. What are they? Non-human species that are used in the laboratory to study biological processes. Each model organism provides unique advantages. Characteristics to consider when choosing a model organism What makes a good model organism? Size Generation time: fast development Embryo accessibility Genome sequenced Haploid Amenable to genetic/molecular analysis Closely related to humans: contains orthologues of genes important for development in humans Mice Mice have an evolutionarily close relationship with humans. Advantages: They have complex biological systems that resemble those in humans (immune, endocrine, nervous, etc.). The protein coding regions of the mouse and human genomes are around 85% identical. Why don’t we always use them to study development and disease? Disadvantages: Genetic manipulation is harder than in other model organisms (creating mutant mice). Expensive (mice need more supervision and maintenance than worms or flies). Ethical concerns Long generation time - 10 weeks Drosophila One of the most used model organisms in developmental biology research. 6 Nobel prizes have been won for work in Drosophila. Advantages: Small, fast generation time (12 days), large brood size (females can lay 120 transparent eggs per day), genetic manipulation is very easy (gene editing techniques like CRISPR/Cas9 are very efficient), easy and cheap to maintain in the lab and grow at room temperature Disadvantages: Anatomy of the brain and other major organs are very different from humans, can’t study behavior, disease states often can’t be modeled in flies, only 50% of protein coding genes are shared with humans C. elegans C. elegans is non-parasitic nematode. Used as a model system in nearly every field of biology research including development. Advantages: Small (1mm), fast generation time (3.5 days), large brood size (140 eggs per day), they are self-fertilizing hermaphrodites (no need to cross to maintain strains), transparent, genetic manipulation is very easy, cheap and easy to maintain in the lab and grow at room temperature, strains can be frozen for many years Disadvantages: Simple anatomy compared to flies, mice, and humans Lacks a major epigenetic mark present in humans that is important in development (DNA methylation) Sea urchin Female sea urchins can be induced to shed their gametes by The sea urchin Strongylocentrotus injection of 0.5M KCl purpuratus has been used as a into the body cavity. model organism for over 150 years. Advantages: Easy to propagate in the lab, the embryo is transparent and has a simple structure, rapid embryogenesis (1-2 days from fertilization to larva), easy to get many synchronized embryos at once. Sea urchin development may illuminate features of the developmental program of the last common ancestor of modern deuterostomes. Sea urchin Disadvantages: Adult animals require a relatively large amount of laboratory space, the time between generations can be quite long (several months) as the reproductive season is limited, sea urchins cannot be inbred so the animals studied are genetically polymorphic Which model organism am I? Because I am transparent, the fate of every cell can be followed throughout development (from the embryo to the adult). I am the best choice to study possible causes or treatments of most human developmental disabilities such as autism spectrum disorder. I’m a great choice for a new lab that doesn’t have that many resources (including space) but wishes to study the impacts of a specific gene mutation in males. I’m working with a PhD student who is trying to figure out how sperm can find eggs to fertilize in a space as vast as the ocean. Animal life cycle Development of the leopard frog Rana pipiens: 1. Fertilization. 2. Cleavage. 3. Gastrulation and development of the body axes. 4. Organogenesis 5. Maturation. 6. Gametogenesis. Fertilization Fusion of the mature gametes. The male and female pronuclei (which are both haploid) fuse to give the embryo its genome. A fertilized egg cell is called a zygote. Cleavage Cleavage is a series of mitotic divisions that follow fertilization. Different organisms undergo cleavage in different ways. Blastomeres Blastula During cleavage, the size and volume of the embryo remains the same, but the blastomeres become smaller. Gastrulation Gastrulation involves a massive amount of cell movements, cell growth, cell divisions, cell shape changes, and cell death. The end result of gastrulation is the creation of three primary germ layers: endoderm, mesoderm, and ectoderm. Ectoderm Mesoderm Endoderm  *This figure does not depict all tissues, only a few representative ones. All three germ layers communicate with each other to form organs and tissues. Body axis establishment Studying development Fate mapping Which cells in the Drosophila embryo form the eyes? Which cells in the mouse embryo form the heart? To answer the above questions, we need to construct a fate map. Fate map: graphical representation detailing the fate of each part of an embryo. We need a way to observe development. Observing embryos – direct observation C. elegans embryos are clear and the embryonic cell lineages were mapped through light microscopy by John Sulston in 1983. Sulston et al. Developmental Biology (1983) Observing embryos – direct observation The fate of cells in tunicate Styela partita were mapped by Edwin Conklin in 1905 through direct observation because of its differently colored cytoplasms. The blastomeres that form the muscle have a yellow color. Observing embryos – vital dyes Dyed agar chips were placed on different parts of the amphibian embryo. The dye diffused into the tissue and the agar was removed. Disadvantage: the dye becomes diluted with each cell division and becomes more difficult to detect over time. Observing embryos – fluorescent dyes Fluorescent dye can be injected into specific cells in the early embryo. Fluorescent dye will also become diluted over time but because it is so much more intense it will be visible for many generations. Observing embryos – genetic labeling Chimeras of two different species A chimera is an organism containing a mixture of genetically different tissues, formed by experimental manipulation.  Observing embryos – genetic labeling Chimeras of two different species A chimera is an organism containing a mixture of genetically different tissues, formed by experimental manipulation. It is difficult to generate chimeras from most species. One way to circumvent this issue is to use fluorescent The grafted quail cells markers such as GFP (green became the wing fluorescent protein). feather cells. This means that cells in that location of the embryo develop into wing feathers. Observing embryos – transgenic DNA chimeras A. A chick embryo was generated that expresses GFP in all tissues B. A region of the neural tube is excised from the GFP chick and transplanted into the same region in the non-GFP chick C. The green neural crest cells are migrating from the neural tube to the stomach region D. Four days later, the neural crest cells have spread in the gut from the esophagus to the anterior end of the hindgut Fate mapping with transgenic DNA showed that the neural crest cells are critical in making the gut neurons. Creating animals that express GFP Transgene: a gene which is artificially introduced into an organism Transgenes are typically introduced into mammalian cells in the form of plasmids Plasmids are small, circular, double-stranded DNA molecules that are distinct from a cell’s chromosomal DNA. Plasmids can express any gene you want. In this example, the plasmid is expressing GFP alone. transcription + translation of the promoter transgene transfection plasmid GFP Cell Cell expressing GFP Creating animals that express GFP Transgene: a gene which is artificially introduced into an organism Transgenes are typically introduced into mammalian cells in the form of plasmids Plasmids are small, circular, double-stranded DNA molecules that are distinct from a cell’s chromosomal DNA. Plasmids can express any gene you want. In this example, the plasmid is expressing GFP alone. promoter GFP translation plasmid GFP Protein of interest Gene of interest Creating animals that express GFP Example: you want to create a fly that expresses GFP in the wings wing- specific- promoter translation plasmid GFP Gene of Thuma et al. 2018 interest Problem You are a developmental biologist trying to understand why certain human babies are born with limb differences (e.g. missing fingers, one arm is bigger than the other, etc.). You have a large lab and enough funds (money) to do whatever you want. To begin your research, you must first create a fate map of a model organism. 1. Pick a model organism and explain why you have chosen that one. 2. Pick a technique to create a fate map and explain why you have chosen that one. Lecture 2: Studying development + cell specification  Observing embryos – genetic labeling Chimeras of two different species A chimera is an organism containing a mixture of genetically different tissues, formed by experimental manipulation. It is difficult to generate chimeras from most species. One way to circumvent this issue is to use fluorescent The grafted quail cells markers such as GFP (green became the wing fluorescent protein). feather cells. This means that cells in that location of the embryo develop into wing feathers. Observing embryos – transgenic DNA chimeras A. A chick embryo was generated that expresses GFP in all tissues B. A region of the neural tube is excised from the GFP chick and transplanted into the same region in the non-GFP chick C. The green neural crest cells are migrating from the neural tube to the stomach region D. Four days later, the neural crest cells have spread in the gut from the esophagus to the anterior end of the hindgut Fate mapping with transgenic DNA showed that the neural crest cells are critical in making the gut neurons. Creating animals that express GFP Transgene: a gene which is artificially introduced into an organism Transgenes are typically introduced into mammalian cells in the form of plasmids Plasmids are small, circular, double-stranded DNA molecules that are distinct from a cell’s chromosomal DNA. Plasmids can express any gene you want. In this example, the plasmid is expressing GFP alone. transcription + translation of the promoter transgene transfection plasmid GFP Cell Cell expressing GFP Creating animals that express GFP Transgene: a gene which is artificially introduced into an organism Transgenes are typically introduced into mammalian cells in the form of plasmids Plasmids are small, circular, double-stranded DNA molecules that are distinct from a cell’s chromosomal DNA. Plasmids can express any gene you want. In this example, the plasmid is expressing GFP alone. promoter GFP translation plasmid GFP Protein of interest Gene of interest Creating animals that express GFP Example: you want to create a fly that expresses GFP in the wings wing- specific- promoter translation plasmid GFP Gene of Thuma et al. 2018 interest Problem You are a developmental biologist trying to understand why certain human babies are born with limb differences (e.g. missing fingers, one arm is bigger than the other, etc.). You have a large lab and enough funds (money) to do whatever you want. To begin your research, you must first create a fate map of a model organism. 1. Pick a model organism and explain why you have chosen that one. 2. Pick a technique to create a fate map and explain why you have chosen that one. Cell specification Textbook Chapter 2 Pages 35 – 41, 43 – 45 To get from a simple, unorganized egg to a complex organism, cells must differentiate into unique cell types Red blood Skin cells cells Smooth Neurons muscle cells Chapter overview Undifferentiated embryo cells go through a series of maturation steps in their journey from a single celled zygote to a fully differentiated cell. In some organisms, cell fate is determined very early in embryogenesis. In other organisms, cell fate remains flexible in the early embryo and becomes fixed through cell-to-cell interactions.  Cell specification maturation steps The generation of specialized cell types (cardiomyocytes, red blood cells, neurons, etc.) is called differentiation. This is the process by which an undifferentiated embryo cell develops specialized structure elements and distinct functional properties. Differentiated cells acquire unique gene expression pattern. Differentiation Undifferentiated cell Differentiated cell  Cell specification maturation steps Differentiation is preceded by a process called commitment. Commitment is the assignment of cell fate, and cell differentiation is the morphological changes in the cells that makes them specialized. Commitment Differentiation 1) Specification 2) Determination (reversible) (irreversible) Undifferentiated cell Committed cell Differentiated cell  Cell specification maturation steps Two stages in cell commitment: specification + determination During the course of commitment, a cell might not look different from its neighbors in the embryo, or show any visible signs of differentiation, but its developmental fate has become restricted. It is during differentiation that cells acquire differences in shape, internal structure, biochemical pathways. Commitment Differentiation 1) Specification 2) Determination (reversible) (irreversible) Undifferentiated cell Committed cell Differentiated cell Stage 1 of commitment: Specification The fate of a cell is specified when it is capable of differentiating autonomously (i.e. by itself) in a neutral environment (such as a petri dish) just as it would inside the embryo (becomes what would be expected based on the fate map). Specification is labile, meaning it can be reversible. If specified cells are placed in a non-neutral environment, such as a population of differently specified cells, the fate of the transplanted cell will be altered by its neighbors.  Stage 1 of commitment: Specification Neutral environment The cells have acquired some signals that specified them to become either muscle cells or neuron cells. When allowed to continue maturing in isolation they will differentiate into muscle and neuron cells.  Stage 1 of commitment: Specification Neutral environment Non-neutral environment The cells have acquired some When a specified signals that specified muscle cell is them to become either transplanted in the muscle cells or neuron middle of a population cells. When allowed to of specified neurons, continue maturing in the specified muscle isolation they will cell becomes a differentiate into neuron. muscle and neuron cells. Stage 2 of commitment: Determination A cell is said to be determined when it is capable of differentiating autonomously even when placed into a different region of the embryo or into a cluster of differently specific cells in a petri dish. Commitment is irreversible at this point. Stage 2 of commitment: Determination Muscle cell was determined Muscle cell was specified but not yet determined  You are a new student in a lab. You’ve been given a project that is to try to figure out when the mouse cells that are bound to become muscle cells are determined in the mouse embryo. You are given a fate map by your supervisor. Hypothesis: on day 14 of mouse embryogenesis, the cells that will become muscle cells are determined. Describe an experiment that will test your hypothesis. Three modes of specification How does an embryo learn what its fate is supposed to be? Autonomous specification The cell know its fate without having to interact with other cells. Cell fate determination is earlier in embryo development. Conditional specification The cell learns its fate by interacting with other cells. Cell fate determination is later in embryo development. Syncytial specification Important in insect development, uses elements of both autonomous and conditional specification. Nuclei divide in a shared cytoplasm. No embryo uses only autonomous, conditional, or syncytial mechanisms to specify its cells.  Commitment Differentiation 1) Specification 2) Determination Specification: the student enters university with an interest in medicine, but they can change their major especially if their peers around them start to express interests in other majors, like engineering. Autonomous specification: the student entered university knowing they were interested in studying medicine without any outside influences Conditional specification: the student was influenced by people around them which made them interested in medicine Determination: the student has entered medical school and will not change their mind. Commitment is irreversible.  Commitment Differentiation 1) Specification 2) Determination Undifferentiated cell Committed cell Differentiated cell Specification: The undifferentiated cell has acquired signals that specify that its fate will be a neuron, but its fate can be changed based on different cues. Reversible. Autonomous specification: the cell knew its fate without having to interact with other cells Conditional specification: cell-cell communications with its neighbors specified its neuronal fate Determination: The cell has been committed to the neuron cell fate. Irreversible. Techniques to study cell specification To study whether cell specification is autonomous or conditional, three major types of experiments are performed: The defect experiment: destroy a portion of the embryo and observe the development of the impaired embryo The isolation experiment: remove a portion of the embryo and observe the development of the partial embryo and the isolated part The transplantation experiment: parts of the embryo are moved around and transplanted onto different embryos or different regions of the same embryo Autonomous specification Autonomous specification is independent of other cells in the embryo. The cell “knows” its fate early on without influence from surrounding cells/tissue. Cell fates are determined early in development. Found often in invertebrate embryos: sea urchin, mollusks, annelids, tunicates Autonomous Transcription factor that specifies muscle cell fate specification Transcription factor that specifies neuron cell fate Cell fate is determined by cytoplasmic determinants found in the blastomeres of the early embryos. These cytoplasmic determinants are often proteins like transcription factors, or mRNA molecules. The cytoplasmic determinants are not homogeneously distributed throughout the egg – different regions of the egg contain different determinants.  Transcription factor that specifies muscle cell fate Transcription factor that specifies neuron cell fate This cell has been specified to become a muscle cell This cell has been specified to become a neuron cell Unequal partitioning of cytoplasmic determinants ensures that during mitosis, each cell receives a unique set of proteins/mRNAs. Autonomous specification – snail How to determine if a cell specifies autonomously? Isolation experiment: Remove early blastomeres and isolate them in a petri dish to see if they develop into differentiated tissues autonomously. These early blastomeres have already been specified and determined to become trochoblast cells. Autonomous specification – tunicate Styela partita How are the B4.1 cells specified to become muscle cells? Autonomous specification – tunicate Styela partita Isolation experiment (figure to the right): when the four blastomere pairs of the 8-cell embryo are isolated, each forms the structures it would have formed in the embryo. This provides evidence that all four blastomere pairs are specified autonomously. Defect experiment (not depicted here): when the B4.1 cells are removed from the 8-cell embryo, the larva develops with no tail muscles. This indicates that only B4.1 cells have the capacity to become tail muscle cells and provides more evidence that these cells are autonomously specified. Autonomous specification – tunicate Styela partita 30 years after experiments showed that the B4.1 cells autonomously specify into tail muscle cells, a scientist named J.R. Whittaker discovered the cytoplasmic determinant responsible for the specification. mRNA for a muscle-specific transcription factor called Macho is found in the yellow B4.1 cytoplasm. Autonomous specification – tunicate Styela partita Loss of macho mRNA leads to a loss of muscle differentiation in B4.1 cells, and injection of macho mRNA into other cells leads to ectopic muscle differentiation. How can you see mRNA in an embryo or a cell? In situ hybridization is a technique that allows you to visualize the location of RNA within cells. Labeled DNA probes (nowadays most labeled are with fluorescent dyes or proteins) bind complementary RNA targets.  Autonomous specification – frogs Another experiment that was performed to test if cells are specified autonomously was Wilhelm Roux’s experiments on frog embryos. Defect experiment: When he killed one of the cells in a 2-cell embryo, the other half of the larva developed normally. This shows that the specification is autonomous (each cell in the 2-cell embryo contained cytoplasmic determinants that specified their fates). Testing the hypothesis that during the first cleavage event there would be a separation of left cytoplasmic determinants and right cytoplasmic determinants. Conditional specification Conditional specification: The fate of a cell depends on interactions with other cells. The early blastomeres of vertebrate embryos are conditionally specified. These interactions include: cell-cell contacts (juxtacrine factors), secreted signals (paracrine factors), and the physical properties of the cell’s environment (mechanical stress). When isolated, the blastomeres can give rise to a wide variety of cell types and sometimes generate cell types that the cell would not normally have made if it were part of the embryo. Blastomeres in the embryo can also change their fate to compensate if parts of the embryo are deleted. Conditional specification Transplantation experiment: If you transplant fate-mapped back cells from the blastula into a region fate-mapped to become the belly, the “back” cells become belly tissue because their environment has changed. Defect experiment: If you remove cells from the dorsal region, the remaining cells can compensate for the missing part. Conditional specification If the back cells were specified autonomously, what would you expect the outcome to be in both experiments?  Conditional specification in the sea urchin Hans Driesch was studying specification in the sea urchin.  Conditional specification in the sea urchin Isolation experiments: Each blastomere from the 4-cell embryo Hans Driesch was regulated its own studying specification development to produce in the sea urchin. a complete organism, rather than self- differentiating into its future embryonic part. Conditional specification in the sea urchin Defect experiment: Driesch also experimentally removed cells, which changed the context for all other remaining cells. This changed the fate of all cells and the embryo developed as normal. Cell fates were changed to suit the conditions. Blastomere biopsy for in vitro fertilization Blastomere biopsies involved the removal of one or two blastomeres from the 8-cell embryo to test for genetic defects. Embryos could develop normally following the removal of one or two blastomeres à conditional specification. Trophoectoderm biopsies are now the most common method of embryo screening. This involves removing cells that will not become part of the fetus but do become part of the placenta. Syncytial specification Syncytium: a cytoplasm that contains many nuclei Syncytial specification: the specification of presumptive cells within a syncytium The identity of cells is established without any membranes separating nuclei into individual cells. Insects are notable examples of embryos that develop through syncytial specification. Syncytial specification in Drosophila For the first 13 cycles of cell division, Drosophila nuclei divide without any cytoplasmic cleavage. Cellularization (formation of membranes) occurs after nuclear cycle 13, just prior to gastrulation. Syncytial specification in Drosophila Many maternal cytoplasmic determinants are loaded into the oocyte/egg. Through mechanisms that we will go over in detail in later lectures, these cytoplasmic determinants form gradients along the anterior-posterior axis. Bicoid Caudal Syncytial specification in Drosophila How are cell fates determined to become the head, thorax, abdomen, and tail before cellularization? 1. Nuclei in the syncytium obtain their identity from their position relative to neighboring nuclei 2. Determination factors are localized in gradients throughout the anterior-posterior axis of the embryo After fertilization, nuclei undergoes synchronous waves of cell division (purple stain). Each nucleus becomes positioned at a specific coordinate along the anterior-posterior axis and experiences a unique concentration of determination factors. How do nuclei maintain their position? In between nuclei divisions (interphase), each nucleus radiates microtubules that are organized by centrosomes. These microtubules establish an orbit and exert force on the orbits of other nuclei, keeping them all in place. Each time nuclei divide, the radial microtubules are reestablished to ensure even spacing between all nuclei. Gradients of determination factors Expression of Bicoid protein Each nucleus is exposed to different amounts of determination factors which are expressed as a gradient across the embryo. Bicoid and Caudal are two transcription factors that set up the anterior-posterior axis. High concentration of Bicoid at the anterior end à head High concentrations of Caudal at the posterior end à tail Medium amounts of both in the middle à thorax/abdomen Problem What experiments could you perform to determine what mode of specification A.1 uses? Explain the results you would expect for autonomous vs. conditional specification. Cell specification (continued) Three modes of specification (Recap) How does an embryo learn what its fate is supposed to be? Autonomous specification The cell know its fate without having to interact with other cells. Cell fate determination is earlier in embryo development. Conditional specification The cell learns its fate by interacting with other cells. Cell fate determination is later in embryo development. Syncytial specification Important in insect development, uses elements of both autonomous and conditional specification. Nuclei divide in a shared cytoplasm. No embryo uses only autonomous, conditional, or syncytial mechanisms to specify its cells. Techniques to study cell specification (Recap) To study whether cell specification is autonomous or conditional, three major types of experiments are performed: The defect experiment: destroy a portion of the embryo and observe the development of the impaired embryo The isolation experiment: remove a portion of the embryo and observe the development of the partial embryo and the isolated part The transplantation experiment: parts of the embryo are moved around and transplanted onto different embryos or different regions of the same embryo Syncytial specification Syncytium: a cytoplasm that contains many nuclei Syncytial specification: the specification of presumptive cells within a syncytium The identity of cells is established without any membranes separating nuclei into individual cells. Insects are notable examples of embryos that develop through syncytial specification. Syncytial specification in Drosophila For the first 13 cycles of cell division, Drosophila nuclei divide without any cytoplasmic cleavage. Cellularization (formation of membranes) occurs after nuclear cycle 13, just prior to gastrulation. Syncytial specification in Drosophila Many maternal cytoplasmic determinants are loaded into the oocyte/egg. Through mechanisms that we will go over in detail in later lectures, these cytoplasmic determinants form gradients along the anterior-posterior axis. Bicoid Caudal Syncytial specification in Drosophila How are cell fates determined to become the head, thorax, abdomen, and tail before cellularization? 1. Nuclei in the syncytium obtain their identity from their position relative to neighboring nuclei 2. Determination factors are localized in gradients throughout the anterior-posterior axis of the embryo After fertilization, nuclei undergoes synchronous waves of cell division (purple stain). Each nucleus becomes positioned at a specific coordinate along the anterior-posterior axis and experiences a unique concentration of determination factors. How do nuclei maintain their position? In between nuclei divisions (interphase), each nucleus radiates microtubules that are organized by centrosomes. These microtubules establish an orbit and exert force on the orbits of other nuclei, keeping them all in place. Each time nuclei divide, the radial microtubules are re-established to ensure even spacing between all nuclei. Gradients of determination factors Expression of Bicoid protein Each nucleus is exposed to different amounts of determination factors which are expressed as a gradient across the embryo. Bicoid and Caudal are two transcription factors that set up the anterior-posterior axis. High concentration of Bicoid at the anterior end à head High concentrations of Caudal at the posterior end à tail Medium amounts of both in the middle à thorax/abdomen Problem If you destroy 1 cell in a 2-cell embryo and the entire embryo develops normally, what concept is this demonstrating? A. Syncytial specification B. Autonomous specification C. Conditional specification D. Differentiation Problem What experiments could you perform to determine what mode of specification A.1 uses? Explain the results you would expect for autonomous vs. conditional specification. Differential Gene Expression Textbook Chapter 3 – whole chapter Chapter overview Genomic equivalence Review of gene expression + central dogma Enhancers + silencers Transcription factors Mechanisms of differential gene expression All multicellular organisms begin as one cell A series of mitotic divisions lead to the generation of a multicellular organism from a single-celled zygote. This means every single cell in the body has the exact same DNA (mitosis produces daughter cells that are genetically identical). Genomic equivalence The concept that every somatic cell in the body has the same chromosomes and set of genes as every other somatic cell in the body. How do different cell types express different sets of proteins and have different physical characteristics if they all have the same DNA? Differential gene expression Differential gene expression is the process by which cells become different from one another by expressing unique combinations of genes. Three postulates of differential gene expression: 1. The DNA of all somatic cells of an organism contain the complete genome that was established in the fertilized egg. 2. The unused genes in each cell are not destroyed and they retain the potential of being expressed. 3. Only a small percentage of the genome is expressed in each cell, and a portion of RNA synthesized in each cell is specific for that cell type. Differential gene expression The genome sequence the identical for all cells in one organism. Which genes would be expressed in all cells? gene coding gene coding for gene coding gene coding gene coding for RNA opsins (proteins for hemoglobin for insulin for ribosomes polymerase II reactive to light) Differential gene expression *don’t need to know these for this course. Just an example of differential gene expression. Some sets of genes are only needed at certain times in development. Evidence for genomic equivalence and differential expression Early experiments in Drosophila. In certain larval tissues, DNA is replicated for several rounds in Drosophila without cell separation This makes it easy to visualize chromosomes by electron microscopy. At different times, different areas of the genome would “puff up”, indicating active transcription and RNA synthesis. This told scientists that not all genes were being transcribed at once, and the activation of certain genes was dependent on the timing of development. Evidence for genomic equivalence and differential expression Differential expression of the odd-skipped gene in Drosophila embryos and mouse embryos. Evidence for genomic equivalence and differential expression Testing genomic equivalence: To test if the genome from a differentiated cell truly has the capacity to generate every other differentiated cell in the body, scientists performed cloning experiments. Genomic equivalence in mammals was proved in 1997 when Dolly the sheep was cloned. Cloning a mammal Can the nucleus of a differentiated cell direct formation of another whole animal? The genome from the isolated udder cells was able to direct the formation of a lamb (Dolly) that was genetically identical to the nuclear donor. This indicated that the genome of udder cells was identical to that of the zygote, and none of the genes necessary to form a whole body are deleted upon differentiation. Dolly with her first born, Bonnie The central dogma The central dogma is the blueprint for how proteins are made in cells. DNA à RNA à protein Gene expression can be regulated at four levels: Transcription (DNA à RNA) pre-mRNA processing (pre-mRNA à mRNA) Translation (RNA à protein) Post-translational modifications (modifications that regulate protein stability and/or function) Chromatin Nucleosome Chromatin In eukaryotes, DNA is wrapped around proteins called histones to form chromatin. The nucleosome is the basic unit of chromatin structure. A nucleosome is composed of a histone octamer (8 proteins – 2 proteins each of histones H2A, H2B, H3, and H4) with 2 loops of DNA wrapped around it (approximately 147 base pairs). Histone tails are positively charged due to the presence of arginine and lysine, which are basic amino acids. The negative charge in the DNA backbone neutralizes the positive charge of histones. Chromatin compaction affects transcription factor binding and gene expression Heterochromatin: tightly packed chromatin regions Euchromatin: loosely packed chromatin regions Anatomy of a gene Promoter: RNA polymerase II binds the promoter. Some promoters have a TATA DNA sequence which is bound by the TATA-binding protein (TBP) which helps RNA pol II bind the promoter. Transcription initiation site: Where transcription begins, formation of the 5’ cap on RNA. Exons and introns: Exons are regions of DNA that code for amino acids that will be part of the protein, introns are non-coding regulatory regions that are spliced out of pre-mRNA and are not included in mRNA. Introns must be removed before the mature mRNA exits the nucleus. Anatomy of a gene Translation initiation codon: ATG on the DNA sequence, AUG on RNA. Site where translation of the protein begins. Same ATG codon in every gene. 5’ UTR or leader sequence: UTR = untranslated region. The sequence of base pairs between the transcription initiation start site and the translation initiation site. Often contains regulatory DNA elements. Translation termination codon: TAA, TAG, or TGA on the DNA sequence, UAA, UAG, or UGA on RNA. Signals the end of translation and the ribosome dissociates from the mRNA after encountering this codon. Anatomy of a gene 3’ UTR 3’ UTR: The sequence of base pairs between the translation termination codon and the end of transcription. Contains the polyadenylation site (AATAAA), which is needed for polyA tail insertion. Contains many cis-regulatory elements that determine mRNA stability and translation. Transcription termination sequence: Termination continues beyond the polyadenylation site for about 1000 nucleotides before being terminated. Pre-mRNA processing Series of adenosines (~250 Methylated guanosine placed in in mammalian opposite polarity to RNA itself – no cells) free phosphate Protein coding Promoters and CpG islands In humans and mice, approximately 50-70% of promoters contain CpG islands. CpG: shorthand for a 5’ cytosine followed immediately by a 3’ guanine. The two are linked by phosphate (which links all nucleosides together in DNA). CpG island: regions of DNA with a high frequency of CpG sites. Formal definitions vary, but the usual definition is an observed-to-expected CpG ratio greater than 60% in a region with at least 200bp. CpG island 5’ ACTGA CGCG ATCC CG TCGTA CGCG A CG Gene X 3’ Promoter Other noncoding regulatory elements Enhancers promote gene expression. Dimmer switch – enhancers + silencers Silencers prevent gene expression. On/off switch - promoters Enhancers and silencers Transcription from eukaryotic promoters can be stimulated by enhancers located thousands of base pairs away from the promoter, and even by enhancers on different chromosomes. Silencers are “negative enhancers”. They silence gene expression from promoters. 1 million different enhancer sequences across the human genome. Whether enhancers/silencers are active or inactive depends on if they are bound by the specific transcription factors that recognize those specific DNA sequences. Transcription factors Basal/general transcription factors: group of proteins that bind every promoter in the genome, necessary for transcription to occur. Transcription factors: heterogenous group of proteins that bind specific DNA sequences in enhancers and promoters. The human genome encodes approximately 1500 transcription factors. Transcription factors TF 1 can only bind enhancers that contain “CCGGA”. Each transcription factor binds a specific DNA sequence TF 2 can only bind enhancers that contain “TTAGA”. These small sequences are called motifs. Each enhancer can have multiple motifs to bind multiple TFs at the same time. TF 1 TF 2 TF 2 TF 1 CCGGA TTAGA Enhancer 1 Enhancer 2 Transcription factors (TFs) Transcription factors can activate or repress transcription depending on cellular cues. Enhancers, silencers, and their associated transcription factors control when, where and how a gene is transcribed. Enhancers The human genome contains an estimated 1 million different enhancer sequences. Because of genomic equivalence, every single cell in the body contains the same 1 million different enhancer sequences. Enhancer activation Example: Gene A is a gene in the mouse genome. This gene can be regulated by two enhancers. 1 2 Enhancer activation The two enhancers are tissue-specific. Enhancer 1 will only be activated by transcription factors present in the brain. 1 2 Enhancer activation The two enhancers are tissue-specific. Enhancer 2 will only be activated by transcription factors present in the limbs. 1 2 Enhancer activation Every tissue expresses tissue-specific transcription factors that will activate tissue-specific enhancers. Example of a real gene that is regulated by multiple tissue-specific enhancers:  Identifying enhancers GFP reporter assay: Will this sequence enhance transcription? Reporter: Presumptive enhancer sequence promoter GFP gene Create an embryo that expresses this reporter in every single cell.  Identifying enhancers GFP reporter assay: Will this sequence enhance transcription? Reporter: Presumptive enhancer sequence promoter GFP gene If our reporter contains an enhancer sequence, the reporter gene (GFP) will Can also be used to test become active at when and where an particular times (temporal enhancer is active during expression) and development. places(spatial expression eg tissue specific expression) based on when this enhancers is supposed to be active. Identifying enhancers LacZ gene codes for: LacZ reporter assays: Beta-galactosidase Presumptive enhancer sequence promoter LacZ If beta-galactosidase is transcribed, you will detect enzyme activity in certain tissues (identified by the presence of the color blue) embryos must be fixed before they are exposed to X-gal Mechanisms of differential gene expression Gene expression can be regulated at different levels of the central dogma. Reduced or increased transcription Epigenetic mechanisms (chromatin methylation/acetylation, DNA DNA methylation) Transcription factors RNA Alternative mRNA splicing Differential mRNA longevity Selective inhibition of translation microRNAs Control of mRNA localization protein Post-translational modifications Regulating transcription Transcription can be regulated by many different mechanisms. Two of the major mechanisms in embryonic development are: Epigenetic modifications Control through transcription factors Epigenetic modification Epigenetics: refers to the study of gene expression changes that do not involve changes to the DNA sequence. Examples: histone acetylation or histone methylation of the tails of histones H3 and H4. Acetylation and methylation occur on lysine residues on the histone tails. Histone acetylation – opening chromatin Acetylation: Histone acetyltransferases place acetyl groups on histone tails. Acetyl groups neutralize the positive charge on the histone tails. This loosens DNA and opens the chromatin. Deacetyltransferases remove acetyl groups. Histone methylation – opening or closing chromatin Methylation: histone methyltransferases catalyze the transfer of methyl groups onto histone tails. Demethylases remove histone groups. Depending on which lysine and which histone tail is methylated, histone methylation can enhance or prevent transcription. Example: H3K9me2/3, H3K27me3, and H4K20me3 combined with no acetylation are associated with highly condensed (and silenced) chromatin. H3K4me3 in combination with acetylation is associated with open (and active) chromatin. Lecture 4: Differential Gene Expression Other noncoding regulatory elements (Recap) Enhancers promote gene expression. Dimmer switch – enhancers + silencers Silencers prevent gene expression. On/off switch - promoters Enhancers and silencers (Recap) Transcription from eukaryotic promoters can be stimulated by enhancers located thousands of base pairs away from the promoter, and even by enhancers on different chromosomes. Silencers are “negative enhancers”. They silence gene expression from promoters. 1 million different enhancer sequences across the human genome. Whether enhancers/silencers are active or inactive depends on if they are bound by the specific transcription factors that recognize those specific DNA sequences. Transcription factors (Recap) TF 1 can only bind enhancers that contain “CCGGA”. Each transcription factor binds a specific DNA sequence TF 2 can only bind enhancers that contain “TTAGA”. These small sequences are called motifs. Each enhancer can have multiple motifs to bind multiple TFs at the same time. TF 1 TF 2 TF 2 TF 1 CCGGA TTAGA Enhancer 1 Enhancer 2 Transcription factors (Recap) Basal/general transcription factors: group of proteins that bind every promoter in the genome, necessary for transcription to occur. Transcription factors: heterogenous group of proteins that bind specific DNA sequences in enhancers and promoters. The human genome encodes approximately 1500 transcription factors.  Identifying enhancers GFP reporter assay: Will this sequence enhance transcription? Reporter: Presumptive enhancer sequence promoter GFP gene Create an embryo that expresses this reporter in every single cell.  Identifying enhancers GFP reporter assay: Will this sequence enhance transcription? Reporter: Presumptive enhancer sequence promoter GFP gene If our reporter contains an enhancer sequence, the reporter gene (GFP) will Can also be used to test become active at when and where an particular times (temporal enhancer is active during expression) and places development. (spatial expression eg tissue specific expression) based on when this enhancers is supposed to be active. Identifying enhancers LacZ gene codes for: LacZ reporter assays: Beta-galactosidase Presumptive enhancer sequence promoter LacZ If beta-galactosidase is transcribed, you will detect enzyme activity in certain tissues (identified by the presence of the color blue) embryos must be fixed before they are exposed to X-gal Mechanisms of differential gene expression Gene expression can be regulated at different levels of the central dogma. DNA Reduced or increased transcription Epigenetic mechanisms (chromatin methylation/acetylation, DNA methylation) Transcription factors RNA Alternative mRNA splicing Differential mRNA longevity Selective inhibition of translation microRNAs Control of mRNA localization protein Post-translational modifications Regulating transcription Transcription can be regulated by many different mechanisms. Two of the major mechanisms in embryonic development are: Epigenetic modifications Control through transcription factors Epigenetic modification Epigenetics: refers to the study of gene expression changes that do not involve changes to the DNA sequence. Examples: histone acetylation or histone methylation of the tails of histones H3 and H4. Acetylation and methylation occur on lysine residues on the histone tails. Histone acetylation – opening chromatin Acetylation: Histone acetyltransferases place acetyl groups on histone tails. Acetyl groups neutralize the positive charge on the histone tails. This loosens DNA and opens the chromatin. Deacetyltransferases remove acetyl groups. Histone methylation – opening or closing chromatin Methylation: histone methyltransferases catalyze the transfer of methyl groups onto histone tails. Demethylases remove histone groups. Depending on which lysine and which histone tail is methylated, histone methylation can enhance or prevent transcription. Example: H3K9me2/3, H3K27me3, and H4K20me3 combined with no acetylation are associated with highly condensed (and silenced) chromatin. H3K4me3 in combination with acetylation is associated with open (and active) chromatin. DNA methylation at promoters CpG island 5’ ACTGA CGCG ATCC CG TCGTA CGCG A CG Gene X 3’ Promoter DNA methylation There are two types of promoters based on CpG content: High CpG-content promoters (HCPs): Developmental control genes (genes that regulate synthesis of TFs and regulatory proteins used in the construction of the organism) Default state of these promoters is ON These CpG islands are not usually methylated To be turned off: histone methylation Low CpG-content promoters (LCPs): Genes whose products characterize mature, differentiated cells (e.g. globins of red blood cells, hormones of pancreatic cells) Default state of these promoters is OFF The Cs in these promoters are methylated and this methylation is critical in preventing transcription To be turned on: DNA methylation at the CpG sites is removed DNA methylation – high CpG promoters (HCPs) ß Default ß Addition of repressing histone methyl marks to turn gene off DNA methylation – low CpG promoters (LCPs) ß Active ß Removal of DNA You do not want cell- methylation marks and specific genes to be turned addition of activating histone on in all cells. They must be methyl groups off by default and turned on when necessary. ß Default DNA methylation blocks transcription DNA methylation can block transcription in two ways: Methylation of C can physically prevent TFs from binding DNA Methylated C can recruit proteins that facilitate chromatin modification E.g. MeCP2. MeCP2 recruits histone deacetylases and histone methyltransferases to create heterochromatin. DNA methyltransferases MeCP2 also recruits a DNA methyltransferase called Dnmt3 which catalyzes de novo methylation à spreading DNA methylation patterns are heritable during cell division (mitosis). To maintain DNA methylation during cell division, an enzyme called Dnmt1 recognizes the methylated cytosine on the parental strand of DNA and methylates the daughter strand. DNA methyltransferases 5’- CGAGCCGTAGCG -3’ 3’- GCTCGGCATCGC -5’ DNA methyltransferases 5’- CGAGCCGTAGCG -3’ 3’- GCTCGGCATCGC -5’ Dnmt1 5’- CGAGCCGTAGCG -3’ 3’- GCTCGGCATCGC -5’ Transcription factors Regulate gene expression by: Recruiting histone modifying enzymes E.g. Pax7 recruits the histone methyltransferase Trithorax complex to methylate H3K4, resulting in transcription activation Stabilizing RNA polymerase activity (+ basal transcription factors) E.g. MyoD stabilizes a basal TF called TFIIB, which supports RNA polymerase II at the promoter Coordinating the timely expression of multiple genes E.g. Pax6 binds enhancers for multiple genes that are supposed to be expressed in the lens cells to activate simultaneous expression of those lens genes Control of gene expression can be positive or negative.  The carboxyl termini are thought to be the trans-activating domains Transcription factors TFs contain 3 main domains: DNA binding domain Trans-activation domain Protein-protein interaction domain A domain is a region of the protein’s polypeptide chain that forms independently from the rest. They are the structural and functional units of proteins. MITF transcription factor à Pioneer transcription factors When transcription factors bind enhancers and loop towards promoters, they can open the chromatin near the transcription start site, allowing pol II to bind. But how do transcription factors bind enhancers if the enhancers are covered by nucleosomes? Pioneer transcription factors They are the first to begin the process of making a locus available for transcription because they can bind and open heterochromatin. Mayran and Drouin. 2018. Journal of Biological Chemistry. Pioneer transcription factors shape the epigenetic landscape. Gene Regulatory Network (GRN) Pioneer TFs begin the process of differentiation; they are not sufficient to complete the process. Different cell types are created through gene regulatory networks. Mechanisms of differential gene expression Gene expression can be regulated at different levels of the central dogma. Reduced or increased transcription Epigenetic mechanisms (chromatin methylation/acetylation, DNA DNA methylation) Transcription factors RNA Alternative mRNA splicing Differential mRNA longevity Selective inhibition of translation microRNAs Control of mRNA localization protein Post-translational modifications Mechanism of differential gene expression: pre-mRNA processing To be translated, once transcribed a pre-mRNA molecule translation must be: Spliced in the nucleus to remove introns Translocated from the nucleus to the cytoplasm Alternative splicing: a process that allows for a single gene to translation form more than one protein  Alternative splicing allows different proteins to be made from the same gene. Mechanism of differential gene expression: pre-mRNA processing All introns have a 5’ splice site and a 3’ splice site. These splice sites are recognized by a multi-subunit protein/RNA complex called the spliceosome. Mechanism of differential gene expression: pre-mRNA processing The spliceosome cuts out the sequence in between the intron between the “GU” and the “AG”.  Mechanism of differential gene expression: pre-mRNA processing ISS – intronic splicing silencer hnRNP - bind splicing silencers to ISE – intronic splicing enhancer inhibit splicing ESE – exonic splicing enhancer SR proteins – bind splicing ESS – exonic splicing silencer enhancers to promote splicing Kornblihtt et al. 2013 Mechanism of differential gene expression: pre-mRNA processing  Cell type-specific proteins can promote or inhibit splicing at specific introns and exons, leading to alternative splicing. Dscam: a gene that can produce 38,016 different proteins by alternative splicing Dscam contains 115 exons. Each neuron expresses a different isoform. When two dendrites from the same Dscam-expressing neuron come in contact, they repel each other. Ensures that dendrites from the same neuron don’t form synapses on each other. Mechanism of differential gene expression: mRNA translation Once the mRNA has been efficiently processed (spliced, capped, polyadenylated) it will be exported for translation in the cytoplasm. The rate of translation is another mechanism by which cells can control gene expression. mRNA stability mRNA accessibility (translatability) mRNA stability – half-life The longer an mRNA is stable in the cytoplasm, the more protein will be translated from it. Half-life: the time required for degradation of 50% of the existing RNA molecules Half-life is measured by using a molecule called Actinomycin D which stops transcription. You then measure how long it takes for all the mRNA molecules of a given gene to be degraded. Sequences in the 3’UTR recruit either destabilizing or stabilizing RNA-binding proteins (RBP) that dictate the half-life of the mRNA. mRNA translatability - stored oocyte mRNAs Eggs/oocytes are often filled with mRNA. This is Oocyte called maternal mRNA. This maternal mRNA in the oocyte is kept translationally inactive until fertilization. Example of translation inhibition in the early embryo: The oocyte of the tobacco hornworm moth produces some uncapped mRNAs that cannot be translated. A methyltransferase adds the 5’ cap upon fertilization, and mRNA translation can proceed. It is the maternal mRNA that provides instructions for early developmental events after fertilization has occurred. Stored maternal mRNAs In zebrafish futile mutants, the female and male pronuclei do not fuse. Despite not having a functional zygotic genome, early cleavage processes progress perfectly well due to the presence of maternal mRNAs that can dictate cell divisions.  mRNA stability and translatability – microRNAs miRNA 3’UTR miRNAs: Small RNA molecules Bind complementary sites in the 3’ UTR of mRNAs (not Blocking Triggering 100% complementary – translation mRNA bulges) initiation degradation > 1000 miRNA sequences in humans, and over 50% of genes contain binding sites for miRNAs in their 3’ UTR microRNAs – lin-4 lin-4 was the first microRNA discovered. Discovered in C. elegans. lin-4 regulates a gene called lin-14. lin-14 has 7 binding sites for the lin-4 microRNA. The LIN-14 protein is a transcription factor that is only needed for the first phase of larval development, the mRNA needs to be quickly degraded after to stop production of the protein to allow the worm to proceed to the next larval stage. lin-4 is responsible for this degradation. microRNA biogenesis The maternal-to-zygotic transition Maternal-to-zygotic switch: For the first few cell divisions after fertilization, all of the proteins produced in the embryo come from the translation of maternal mRNAs. Most genes in the zygote genome are not being transcribed. microRNAs and the maternal-to-zygotic transition Maternal-to-zygotic switch: At a specific time in embryogenesis (different for every organism), the maternal mRNAs are degraded, and the zygotic genome becomes activated. Many mechanisms work together to rapidly degrade maternal mRNAs, including miRNAs. microRNAs and the maternal-to-zygotic transition How are maternal mRNAs degraded? In zebrafish, the microRNA miR430 is responsible. It targets about 40% of the maternal mRNA in the zebrafish embryo. Upto here for quiz 1 Opens Sept 16 at 10: 00 am Closes Sept 20 at 10:00 pm Tools – in situ hybridization What it is: A way to visualize RNA in a cell or embryo. When to use it: You want to know where or when a specific mRNA is expressed in a cell or embryo. Tools – ChIP-sequencing (chromatin immunoprecipitation) What it is: A way to determine the DNA-binding site of specific proteins When to use it: You want to know which enhancers or silencers a specific transcription factor is binding at a given moment during development. You want to know which genes are covered in specific histone modifications (e.g. which genes contain the silencing H3K9me3 mark on day 12 of development) Tools – RNA-sequencing What it is: A way to identify the full repertoire of mRNAs expressed in an embryo, tissue, or a single cell at a given moment. Like taking a snapshot of all mRNAs present in the cytoplasm at a given timepoint. When to use it: You want to ask questions like “which mRNAs are expressed on day 4 of development and how does this compare to the mRNAs expressed on day 9?” You want to know how much of a specific mRNA molecule is present at a given time. Tools – reverse genetics Is gene X necessary for tail development? Remove gene X from embryo à observe development. What is the function of gene Y? Remove gene Y from cell/embryo à observe the cell/embryo Many tools to delete or mutate genes. Mutating a gene to uncover its function – reverse genetics Tools – reverse genetics: RNAi The microRNA pathway can be hijacked by researchers to knock down genes of interest. Small double-stranded RNA molecules called siRNAs (small-interfering RNAs) are introduced into cells and bind complementary mRNA targets and lead to their degradation. This is not permanent as the siRNAs become diluted after several cell divisions, and not 100% of the target mRNA is degraded. Therefore, it’s called a gene knock down. Tools – reverse genetics: CRISPR/Cas9 CRISPR is a naturally occurring anti-viral system in bacteria. When bacteria become exposed to a virus, they integrate a small fragment of that virus into their own genome. When they encounter this virus again, the small DNA fragment is transcribed into a guide RNA (gRNA). The gRNA is bound by an endonuclease called Cas9. When the gRNA binds the viral DNA (through sequence complementarity), Cas9 cleaves the viral DNA generating a double-strand break, disabling the virus. Tools – reverse genetics: CRISPR/Cas9 The double strand break can be repaired by one of two pathways: non-homologous end-joining (NHEJ) and homology directed repair (HDR). NHEJ is an imperfect repair mechanism – often introduces INDELS into DNA. HDR uses a repair template to swap in a fragment of DNA based on homologous sequences between the repair template and the sequences surrounding the double- stranded break. You are a PhD student studying the regulation of a gene called “Florinex-1” (fake gene name) in Drosophila embryos. Your supervisor wants you to determine: a) When and where during embryogenesis it is expressed. b) Which enhancer(s) regulate(s) expression of Florinex-1. There are a number of regions of DNA you suspect may be acting as enhancers: region A, region B, region C. c) This experiment will be performed after you have determined which enhancer activates Florinex-1. If the transcription factor “PEX-1” (that your fellow PhD student is studying) is involved in the activation of an enhancer that regulates the expression of Florinex-1. Describe experiments that will allow you to determine the answers to the above questions. You’re studying the expression of gene Y in zebrafish embryos. You know that protein Y is highly expressed in the developing brain at 60 hours post- fertilization for 2 hours and then expression rapidly drops. By 63 hours post-fertilization there is no protein Y in the brain. Explain four different mechanisms that could have led to a rapid decrease in protein Y expression. Lecture 5: Tools to study developmental biology Tools – in situ hybridization What it is: A way to visualize RNA in a cell or embryo. When to use it: You want to know where or when a specific mRNA is expressed in a cell or embryo. Tools – ChIP-sequencing (chromatin immunoprecipitation) What it is: A way to determine the DNA-binding site of specific proteins When to use it: You want to know which enhancers or silencers a specific transcription factor is binding at a given moment during development. You want to know which genes are covered in specific histone modifications (e.g. which genes contain the silencing H3K9me3 mark on day 12 of development) Tools – RNA-sequencing What it is: A way to identify the full repertoire of mRNAs expressed in an embryo, tissue, or a single cell at a given moment. Like taking a snapshot of all mRNAs present in the cytoplasm at a given timepoint. When to use it: You want to ask questions like “which mRNAs are expressed on day 4 of development and how does this compare to the mRNAs expressed on day 9?” You want to know how much of a specific mRNA molecule is present at a given time. Tools – reverse genetics Is gene X necessary for tail development? Remove gene X from embryo à observe development. What is the function of gene Y? Remove gene Y from cell/embryo à observe the cell/embryo Many tools to delete or mutate genes. Mutating a gene to uncover its function – reverse genetics Tools – reverse genetics: RNAi The microRNA pathway can be hijacked by researchers to knock down genes of interest. Small double-stranded RNA molecules called siRNAs (small-interfering RNAs) are introduced into cells and bind complementary mRNA targets and lead to their degradation. This is not permanent as the siRNAs become diluted after several cell divisions, and not 100% of the target mRNA is degraded. Therefore, it’s called a gene knock down. Tools – reverse genetics: CRISPR/Cas9 CRISPR is a naturally occurring anti-viral system in bacteria. When bacteria become exposed to a virus, they integrate a small fragment of that virus into their own genome. When they encounter this virus again, the small DNA fragment is transcribed into a guide RNA (gRNA). The gRNA is bound by an endonuclease called Cas9. When the gRNA binds the viral DNA (through sequence complementarity), Cas9 cleaves the viral DNA generating a double-strand break, disabling the virus. Tools – reverse genetics: CRISPR/Cas9 The double strand break can be repaired by one of two pathways: non-homologous end-joining (NHEJ) and homology directed repair (HDR). NHEJ is an imperfect repair mechanism – often introduces INDELS into DNA. HDR uses a repair template to swap in a fragment of DNA based on homologous sequences between the repair template and the sequences surrounding the double- stranded break. You are a PhD student studying the regulation of a gene called “Florinex-1” (fake gene name) in Drosophila embryos. Your supervisor wants you to determine: a) When and where during embryogenesis it is expressed. b) Which enhancer(s) regulate(s) expression of Florinex-1. There are a number of regions of DNA you suspect may be acting as enhancers: region A, region B, region C. c) This experiment will be performed after you have determined which enhancer activates Florinex-1. If the transcription factor “PEX-1” (that your fellow PhD student is studying) is involved in the activation of an enhancer that regulates the expression of Florinex-1. Describe experiments that will allow you to determine the answers to the above questions. You’re studying the expression of gene Y in zebrafish embryos. You know that protein Y is highly expressed in the developing brain at 60 hours post- fertilization for 2 hours and then expression rapidly drops. By 63 hours post-fertilization there is no protein Y in the brain. Explain four different mechanisms that could have led to a rapid decrease in protein Y expression. Lecture 5: Cell-to-cell communication Textbook Chapter 4 Pages 87 – 119, 123 – 125, 128 – 129 How do identical-looking blastomeres differentiate into specific cells types (e.g. eye cells, neurons, muscle cells)? How do cells of a certain type know to organize together? How are cells ordered to create tissues? How are tissues ordered to form organs? What keeps the mesoderm separate from the ectoderm? Chapter overview Cells in the developing embryo communicate with each other. Cell adhesion is a factor that dictates the movement of cells and helps physically organize cells within the embryo. Cells adhere to each other + to the extracellular matrix Defining induction and competence: how one tissue can induce a morphological or behavioral change in another tissue Morphogens: a special category of paracrine factor Survey of different paracrine factor families and specific examples of how they function Juxtacrine signaling Communication between cells can be direct or indirect Juxtacrine signaling Communication between neighboring cells in direct contact. Homophilic binding: receptor in the membrane of one cell binds the same type of receptor on another cell. Heterophilic binding: receptor in the membrane of one cell binds a different receptor on the other cell. Could also involve communication between a cell receptor and the extracellular matrix (ECM). Communication between cells can be direct or indirect Paracrine signaling Paracrine factors (also called ligands) are proteins that are secreted from a signaling protein. Ligands bind receptors on neighboring cells receiving the signaling information. Signal transduction cascades Protein-protein interactions (either between ligand + receptor or receptor + receptor or ECM protein + receptor) often lead to a change in protein conformation of the receptor. This change in protein conformation gives the receptor a new property and allows it to relay a signal into the cytoplasm. The series of enzymatic reactions that are triggered by receptor activation is called a signal transduction cascade or signal transduction pathway.  Ectoderm Mesoderm Endoderm Our epidermis comes from the ectoderm. Our dermis comes from the mesoderm. What keeps the ectoderm separate from the mesoderm such that our skin has distinct epidermis and dermis layers? Differential cell affinity Townes and Holfreter in 1955: conducted cell recombination assays to study morphogenesis. Cells reaggregate on their own Amphibian embryonic tissues and become spatially were dissociated into separated. Epidermal cells on individual cells after being outside, neural cells inside. placed in an alkaline solution. These positions reflected their positions in the embryo. Differential cell affinity Townes and Holtfreter continued their experiments. When the three germ layers are mixed together, they sort themselves into positions that reflect their positions in the embryo – endoderm in the middle, separated from the epidermis (ectoderm) by the mesoderm. They attributed these results to the cells exhibiting differential affinity. Differential cell affinity This is also observed when cell types within the same germ layer are recombined (e.g. neural plate + epidermis are both ectodermal cell types). If neural plate cells, mesoderm cells, and epidermis cells are mixed together, they arrange themselves with the neural plate at the center, covered by the mesoderm, covered by the epidermis. Differential adhesion hypothesis Townes and Holtfreter: cell movement during morphogenesis is not random. Something is directing cell movement, but what? 1964: Malcolm Steinberg proposed the differential adhesion hypothesis. “All that is required for sorting to occur is that cell types differ in the strength of their adhesions” Differential adhesion hypothesis The hypothesis proposes that cell segregation arises from tissue surface tensions, which in turn arise from differences in cellular adhesiveness. Cells will interact to form an aggregate with the smallest A group of cells with low surface tension will interfacial free energy. not tend to stick together as much as a group of cells with high surface tension. Low surface tension cells will envelop the high surface tension cells. Differential adhesion hypothesis Cell types with higher surface tension migrate centrally when combined with cells with less surface tension. Dyne/cm is the unit traditionally used to measure surface tension. Differences in surface tension are mainly driven by the presence of different types of adhesion molecules on the surface of cells. Cadherins The major type of cell adhesion molecule is a protein called cadherin (calcium-dependent adhesion molecules). Cadherins bind to cadherins on adjacent cells, keeping them together. They form adherens junctions. Cadherins span the plasma membrane. Inside the cell, they are anchored to actin filaments via catenins. Cadherins perform several important functions: Adherens junction Adherence Connection to the actin cytoskeleton Initiation of signal transduction cascades Cadherins Blocking cadherin synthesis by RNAi or mutations prevents the formation of epithelial tissues and causes cells to disaggregate. Epithelial tissues are highly enriched in cadherins because epithelial cells need to stick together to form tight boundaries. Adherens junction Cadherins Several types of cadherins have been identified in vertebrate embryos: E-cadherin: all early mammalian embryo cells, very important in mediating adhesion of epithelial cells P-cadherin: placenta, helps keep it stuck to the uterus N-cadherin: expressed in cells of the developing nervous system R-cadherin: critical in retina formation Protocadherins: lack the attachment to the actin cytoskeleton inside the cell Surface tension is linearly related to the amounts of cadherin molecules on cell membranes How does the amount of cadherin proteins affect surface tension? Surface tension increases linearly with the amount of cadherins expressed. Green cells have more N-cadherin than red cells.  Surface tension is linearly related to the amounts of cadherin molecules on cell membranes Homotypic aggregate This thermodynamic principle also applies in heterotypic aggregates (when two groups of Heterotypic aggregate cells express different cadherins) E-cadherin in zebrafish In many embryos, the onset of gastrulation is marked by epiboly. Epiboly is a morphogenic movement characterized by tissue spreading and thinning. Typically, the ectoderm cells divide, spread, and become thinner to envelop the entire embryo. Expression of E-cadherin is required to drive this cell spreading and thinning.  Epiboly Epiboly is driven by radial intercalation, a cellular movement that involves cells from deeper layers moving towards the outer layer. This makes the tissue thinner, and it makes the tissue spread. E-cadherin in zebrafish In zebrafish, the inner epiblast cells move outwards into the more superficial epiblast cells. This makes the epiblast thinner and powers tissue spreading. This process is dependent on a high concentration of E-cadherin in the enveloping layer (EVL). The cells of the EVL are anchored there. They cannot move to the center of the embryo despite having high surface tension. Instead, they attract cells towards the more superficial layers of the epiblast, powering radial intercalation. The epiblast cells fail to thin and spread in an E- cadherin mutant (-/-). Extracellular matrix (ECM) ECM ECM: insoluble network of macromolecules secreted by cells. Important for: cell adhesion, cell migration, and the formation of epithelial sheets.  Collagen (the most abundant protein in the ECM) Glycoproteins (oligosaccharides attached to a protein) E.g. fibronect