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DB-Lec 1&2 - Biology 442 Developmental Biology...

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Unformatted text preview: Biology 442 Developmental Biology Biology Biology Lectures 1& 2 – Introduction to Lectures Development: How We Study Development Development All figures in these power points are from either Kalthoff -2ond edition All or Gilbert, 7th or 8th edition Developmental Biology, Sinaur Associates or publisher unless otherwise noted. publisher What is Development ? What Embryology; The study of “how life begins” -- but with the power of Molecular Biology, Genetics and Cell Biology it has become much more encompassing – thus the term “Development” Development is a process which must accomplish 2 functions: 1) Assure the continuity of life from one generation to 1) the next – REPRODUCTION REPRODUCTION 2) Generate the cellular diversity necessary to sustain 2) complex life from one generation to the next -complex DIFFERENTIATION DIFFERENTIATION The study of Development encompasses Growth: Life starts as a single cell – Fertilized Egg Fertilized Growth: Adult Human has 10e14 cells (100 trillion) Adult Most growth involves making MORE cells, not Bigger Cells Most – mitosis – Size of the organism increases. mitosis Differentiation: Fertilized Egg Differentiation: Fertilized Over 200 different cell types ; muscle, skin, neurons, bone …….. different Morphogenesis: Organization of the Morphogenesis: differentiated cells into tissues, organs and the overall body plan. Creation of Form. body Reproduction: – assures the continuity of the species Reproduction: from generation to generation. Asexual – 1 parent – offspring identical Asexual Sexual – 2 parents – offspring are combination of genetic Sexual material from both parents – produce specialized cells for this purpose – gametes – sperm and eggs gametes sperm Evolution: New species evolve via modification of Evolution: existing ones – the study of development provides both confirmation of this and insight to how species evolve. confirmation Major Features of Animal Development Major 2 1 Fertilization – union of sperm and egg to produce the fertilized egg or Zygote The parent sets aside cells – germ cells – that will become the future gametes –egg and sperm 3 Cleavage – rapid mitotic divisions of the fertilized egg. 1) Mechanisms of cleavage, 2) How is cell fate determined ? Are specific cytoplasmic molecules segregated to different daughter cells during cleavage that affect cell fate 3) Do all cells contain the same genetic information ? 4) Setting up the body plan – Axis formation. Gastrulation – Reorganization of the cells of the embryo to generate the 3 germ layers – endoderm, mesoderm and ectoderm that will give rise to the body organ systems. This requires cell migration and changes in cell shape and changes in adhesive properties of cell 4 Neurulation and Axis Formation Ectodermal Organs Mesodermal and Endodermal Organs 5 Limb Formation Anterior-Posterior Pattern Formation Dorsal-Ventral Pattern Formation Cell Differentiation and Cancer Senescence Sex Determination Human Developmental Anomalies Human Developmental Evolution Developmental Historical Background - Where do our ideas about development come from and how is development studied ? how Earliest tool for studying embryogenesis: Observation Observation Aristotle (384-322 BC) – observed development of chickens and other animals whose embryos were large enough to see with the eye Recognized what is a fundamental principle of development -- New structures are generated from preexisting ones -- EPIGENESIS EPIGENESIS 4 day Chicken Embryo Until the advent of better microscopes in the mid 1700s, The “Preformation Theory” was mid was Popular. Popular. “The next generation was prefigured in miniature within the previous generation” Development only involved GROWTH ‘ovists’ and ‘spermists’ – both ‘spermists’ believed that the next generation was just “unrolled” PROBLEMS WITH THIS THEORY !!! Hartsoeker’s Homunculus 1694 Until the advent of better microscopes in the mid 1700s, The “Preformation Theory” was mid was Popular. Popular. discovery of cells puts a size limit on the number of prefigured generations Development only involved GROWTH in one egg or sperm !!! 2) variation ! ‘ovists’ and ‘spermists’ – both ‘spermists’ believednot account for inherited Does that the next generation was just “unrolled” “The next generation was prefigured in 1) How the previous generation” miniature withinsmall is small ? The PROBLEMS WITH THIS THEORY !!! Hartsoeker’s Homunculus 1694 Kaspar Friedrich Wolff - 1767 – looked at chicken eggs Kaspar and saw that developing chick embryos looks nothing like the adult and complex structures come from simple ones. He described blood vessels, guts and kidneys looking different from the adult organs and the neural tube arising from a flat tissue --- consistent with The Principle of Epigenesis Neural plate Neural grove Neural tube With improvements in Microscopy Preformation Ideas gave way to Epigenesis as the principle underlying development What was needed was a way of integrating 3 key facts. integrating 1) Contributions from both parents to the 1) offspring offspring 2) The existence of mutations 2) 3) Epigenesis – each organism develops anew from an undifferentiated condition anew Question – what is the source of instruction Question for the “development of each new organism” organism” Advances in cell biology resulting from better microscopes resulted in the following observations which were central to modern embryology which 1677 – von Leewenhoek sees sperm in semen but says they are little animals that must be parasites of testes 1838 – Schleiden and Schwann propose that all organisms consist of ‘cells’ - cell theory 1841 – Koelliker – sperm is a specialized cell produced in the testis 1861 – Gegenbauer – the egg is one big cell 1870s Hermann Fol and Richard Hertwig observe union of sperm and egg 1870s vanBeneden and Strasburger demonstrate that each somatic cell had a fixed number of chromosomes that was halved (1N) during germ cell (egg and sperm) maturation and doubled (2N) during fertilization. Observation also indicated that from 1 cell (fertilized egg) you get many cell types 1883 August Weismann puts all of these cytological 1883 August observations together with the epigenesis principle to epigenesis formulate The Germ-Plasm Theory Object: to explain how one gets differentiation of many cell types and provides for genetic contributions from both parents and has continuity from generation to generation and is flexible enough to account for mutation. The Germ Plasm Theory The Eggs and Sperm provide equal contributions – they contribute Eggs chromosomes. chromosomes. Chromosomes are the basis for continuity between generations Chromosomes and carry the inherited potential of the organism. and Only nuclei of cells destine to become germ cells (egg +sperm) Only maintain all of the “chromosomal determinants” Somatic cells contain only a fraction of the chromosomal Somatic determinants determinants The Germ Plasm Theory was a major milestone because it provided a testable hypothesis! testable Testing it resulted in the introduction of a new experimental approach – Manipulating embryos: experimental Manipulating To do this you need big easily accessible embryos, tiny tools and steady hands People switched from observing chicken embryos to using tunicates, sea urchins, amphibians, etc 4 basic types of experiments were done: Transplantation experiment: replace one part of Transplantation an embryo with the same part from an “identifiably” different embryo – used for “fate “identifiably” fate mapping”. mapping Isolation experiment: remove a portion of the embryo and observe it develop in isolation (without the rest of the embryo) (without Recombination experiment: replace part of an embryo with tissue from a different region of the embryo. embryo. Defect experiment: destroy a part of the embryo and observe development of impaired embryo and Example :Transplantation experiment: for FATE MAPPING Transplantation for A Proof that the floor plate of the neural tube and the notochord is derived from Hensen’s node. B (A) The node of a 6-somite chick embryo is replaced by its quail counterpart. (B) Analysis of the chimeric axis (stained for the quail-specific antigen), showing quail cells in the notochord (arrow) and floor plate (arrowheads). (B from Catala et al., 1996; photograph courtesy of N. M. Le Douarin.) Using a large number of experiments like this early embryologists Mapped the Fate or Traced the Mapped Lineage of tissues and organs back to individual Lineage cells of cleavage stage embryos cells Embryo Manipulation Experiments Designed to Embryo Test Weismann’s Germ-Plasm Theory Isolation experiment: remove a portion of the Isolation embryo and observe it develop in isolation (without the rest of the embryo) (without Recombination experiment: replace part of an embryo with tissue from a different region of the embryo. embryo. Defect experiment: destroy a part of the embryo and observe development of impaired embryo and Embryo Manipulation Experiments Identified Embryo Major Control Mechanisms Guiding Development Major Defect experiment: destroy a part of the embryo and observe Defect development of impaired embryo development 1888 – Wilhelm Roux collected fertilized Frog’s eggs –let them Wilhelm divide to 2 Cell Stage, killed one cell with a hot needle YES!! if Weismann was right and somatic cells lose determinants then one should only get half the embryo Conclusion: Embryo is a mosaic Conclusion: of self-differentiating parts – Mosaic Development - cells in Mosaic isolation behave like they do in the organism. the Cells have restricted Cells potential – they can only become potential certain tissues or organs certain 1892 Hans Driesch – wanted to understand development in terms of laws of math and physics – “embryo was little machine of self­ differentiating parts” He repeated Roux’s experiment but used sea urchin embryos Sea Urchin embryos too small to kill one cell but he could remove fertilization envelope and disassociate to single blastomeres. Isolation experiment: remove a portion of the embryo and observe it develop in isolation (without the rest of the embryo) (without Each Blastomere gave rise to a whole embryo!!! Was Weismann WRONG ??? The result of his experiment shattered Driesch’s faith in development following laws of math and physics. He left science and took up philosophy!! What he had discovered was What Regulative Development Regulative Each cell retains the developmental potential of the fertilized egg, but only expresses a portion of that potential based on its conditions – conditional specification The part of Weismann’s theory that was wrong was the postulate that somatic cells lost determinants If we think about Weismann’s theory and say instead that somatic cells only EXPRESS some of the determinants then ------Wilhelm Roux’s Mosaic Development represents a situation where the somatic cells are committed to expressing only a subset of determinants Hans Driesch’s Regulative Development represents Hans a situation where the somatic cells are not yet committed to expressing a subset of determinants and are still able to express all determinants if the conditions are right – pluripotent or totipotent Hans Spemann, who later won the Nobel Prize for his research, was also using embryo manipulation experiments to test Weismann’s theory His research uncovered some of the most important concepts in embryology. We will look at 4 of his most important experiments and see how they expand the concepts of mosaic and regulative development and further define the nature of developmental regulation 1903 - Baby Hair Experiment • fertilize a new egg and pull baby’s hair around the middle of it until it is almost closed – nucleus goes to one side – • let divisions happen – all one side • at 16 Cell Stage let a nucleus slips through hole and into other side • tie off lasso and let both sides continue to divide Result: twin embryos, one slightly older than the other! Conclusion: It is the nuclei that contain the potential not the cells and the nuclei could regulate if necessary. Gray Crescent Experiment –done with a salamander embryo that after fertilization has a gray crescent of cytoplasm. A) He split the fertilized egg into 2 cells so that each cell got equal amount of the grey crescent –each cell produced a normal salamander. A) He split the fertilized egg into 2 cells so that only 1 got the grey cytoplasm. Only the cell with the grey cytoplasm produced a normal salamander Result: gray crescent region contained a material which was essential for normal development Conclusion: The nuclei had all determinants, but something in the cytoplasm was needed for normal development . Cytoplasmic Determinant – a factor in the cytoplasm of the egg that gets non-randomly segregated and alters the fate of the cells that get it 1918 - commitment experiment From the fate map of the frog embryo he knew that one part of embryo became dorsal nervous system structures and another part became ventral skin Experiment: At early gastrula take tissue that would become neural structure and transplant it in another early gastrula embryo to a hole where he removed epidermally –fated tissue Result – it became what the cells he removed would have become – Epidermis. Conclusion: cells could change their fate and adopt the fate of cells at the new site. This established the influence of location on differentiation Next he repeated the experiment with cells from late gastrula but transplanted them into an early gastrula embryo He got a different result !! – The cells became what they would have if they had stayed in their original location – they became neural tissue but on ventral side where epidermis should have formed. LESSON – cells from the later stage of embryogenesis were committed to their cell fate irreversibly Thus developmental timing is important – cells lose potential with time potential Induction Experiment done by Ventral side Dorsal side Hilde Mangold a graduate student Hilde of Spemann’s -- Used newt embryos – just prior to gastrulation there is a very important group of cells that form at a part of the embryo known as the “dorsal blastopore lip” (DBL). This is where gastrulation initiates. She took cells from DBL of a dark species of newt and transplanted them to the ventral side of an early stage embryo from a light species of newt. When she had done this with other early tissues they adopted fate of the ventral side cells that they replaced. The DBL didn’t do that – it did what it would have in its normal spot – it formed a blastopore on ventral side and cells started moving inside on both sides– They went on to make all the normal dorsal structures, neural tube, notochord, backbone and gut –they got embryos with a duplicated embryonic axis or a secondary axis – When looked at the dorsal tissues that formed, they weren’t all dark – the light cells from the recipient had also contributed to these structures The dark DBL tissue had altered the fate of the cells it was placed next to !! The DBL was different – it could change the cells around it. This process of one cell or group of cells telling other cells what to become is called Induction Induction The job of DBL cells is to induce other cells in embryo to form neural tube, notochord – dorsal structures – this area of embryo is called the Spemann Organizer Spemann he called this primary embryonic induction primary From these simple tools – 1) Observation, first with the eye Observation first alone and then with microscopes and 2) Embryo Manipulation Embryo - an enormous amount was learned prior to the mid 1900s. an Sperm/egg fusion to form the fertilized embryo was established. Mosaic development, regulative development cytoplasmic determinants, commitment to a particular fate and induction were all established. But how does this happen? - What molecules are involved? – One of big objectives of Developmental Biology research has been to understand the molecular mechanisms underlying the function of Spemann’s organizer. The identification of the molecules involved in all of these processes are now being identified by genetics and molecular biology Genetic Approaches to studying Development. Development. Gene — fundamental unit of heredity — usually, but not Gene always, makes a protein — genes control a character or trait or process A change in gene is called a mutation - mutations can mutation be dominant, recessive, heterozygous, homozygous. dominant, Genotype – the genetic makeup of an organism Phenotype – the visible characteristics of an organism Phenotype Genetic approach to Development: Identify Genetic a process and look for mutants in which the process is defective. The mutations will identify genes whose products function in the process. Types of mutant alleles and types of mutations: 1. Null allele – complete loss of a gene or its function. complete 2. Loss of function allele – some but not all of a gene’s function is lost. Most mutants are of this type. function 3. Gain of function allele – a gene becomes active at a time or in a place where it is normally silent. It can also be an overexpressing mutant. 4. Lethal – kills the organism 4. kills 5. Temperature sensitive (conditional) - get phenotype only at certain temperatures—usually due to disrupting function of protein at higher temperatures. function permissive temperature—things are normal restrictive temperature—things are mutant —can shift at —things different times and see when gene is needed different In Development there are 2 major classes of mutations – Maternal effects classes Maternal mutations and Zygotic mutations mutations Zygotic Maternal genes make products that are stored in the egg and Maternal used during early development. 2 types of maternal genes: Housekeeping – used in all cells, histones, tubulin. Cytoplasmic determinants – products which specify a fate on cells that inherit them. Zygotic genes - genes from the Zygotic zygote nucleus expressed at blastula stage and through the rest of life Maternal effects mutations are mutants of the mother that Maternal influence the phenotype of the offspring but not necessarily its genotype Example of a maternal effects mutation mutation Bicoid – a protein made by the mother and put in egg cytoplasm and located in a gradient from anterior to posterior in the developing embryo. Offspring could have a wild type bicoid gene from father but it would not help!! Zygotic mutations can also have dramatic effects on development. on Ultrabithorax – wings in T3 segment instead of halteres -- T2 replaces T3 because of a loss of Legs not function mutation in Ubx Antenna !! Zygotic loss of function mutation. mutation Zygotic gain of function mutation (a) Head of a Wild-type Fruit Fly. (B) (a) Antennapedia gene expressed in head Head of a Fly Containing the Antennapedia Mutation Antennapedia Saturation Mutagenesis Screens: Used for the Saturation Used genetic analysis of biological processes, they aim to generate mutations in nearly all genes involved nearly in the process of interest. in How do you know that you have mutated every gene that functions in a process ? A Poisson distribution (statistical calculation) calculates that an average of 5 mutant alleles per gene must be obtained in order to have a 99% chance of mutating every gene in the process. This probability analysis tells you how many mutants you need to screen. If there are 5000 genes predicted to control development and your mutagenesis procedure generates 0.7 mutations per treated genome then the number of lines that you need to screen for 99% saturation is 5000X5/0.7=36,000 mutants. Saturation mutagenesis screens have been used by Saturation Nusslein-Volhard and Wieschaus to generate mutations in every gene in Drosophila that is involved in laying down the body axes. They won the Nobel Prize for this in 1995 together with Lewis who discovered the first homeotic mutations by classical genetics. They have been used now to identify major pattern formation genes in Zebra Fish as well. What does one do with the mutants that one has isolated ? 1) Characterize the phenotype and 1) group the genes based on phenotype 3) Complementation analysis to determine if two mutations, Complementation often with similar phenotypes are in the same or different genes --- allelic means different mutations in the same allelic gene. 1 each mutant is crossed with wild type to 2ond heterozygotes of each mutant are crossed to each other. If all of the offspring of these crosses have a wild type phenotype then the mutations are in 2 different genes and phenotype each parent in the cross brings in a wild type copy of the other gene. If the cross produces offspring that do not show the wild phenotype, the mutated genes fail to complement and are allelic. Once you have your mutants – you Once want to know what gene is mutated and this involves Molecular Biology!! and Organisms that are good for studying development using genetics – using drosophila, worms, zebra fish and mice mice Molecular Approaches Molecular From genetic analysis -- get mutants, map them, determine how many genes are essential for a pathway and order the genes into a hierarchy of function but now we want to know what the gene is or what it does or in what tissues or when during development it is made. For this we need Molecular Biology. Biology The genomes of many important developmental organisms have been sequenced. These include worms, drosophila, dictyostelium, mice, humans, arabidopsis You know where your gene maps so you look at genes that are located in the region of the genome where your mutant maps. Do any encode proteins whose mutation could account for your mutant phenotype ? Suppose you have isolated a mutant in which cells can not migrate. You have named your mutant stuck in place (sip). We know cadherins and integrin receptors are required for motility so we ask do genes like that map where my mutant maps ? This is known as the candidate gene approach This is how many human disease genes are identified But we need to know for sure that the candidate gene is the one mutated in sip. Two approaches. (1) in flies, worms, dictyostelium, zebra fish—get pieces of DNA from area of chromosome around where gene lies and inject them into mutant animals and see if any now give wildtype progeny back— have rescued the mutant and cloned your have gene (2) take DNA from your mutant and sequence the region where the mutation is and look for changes in DNA sequence in region Once you have identified your gene and have a clone of it you can ask when during development is it expressed and in what tissues. Several ways are used. 1. Northern blots 1. In situ hybridization 2. Use of reporter genes In situ hybridization In Chicken embryo showing staining from in situ hybridization in tissues that express “your mRNA” Reporter gene expression: Make a plasmid containing the control regions of your gene (Sip) fused to a reporter gene (b-gal or GFP). The plasmid needs to contain a selectable marker. Promoter for sip gene Coding region of sip gene replaced with gene encoding E. coli bgalactosidase enzyme Gene encoding a selectable marker – G418 Transform or microinject your plasmid DNA into cells or into a early embryo, let the cells or embryo grow and then harvest, fix and permeablize and add chromogenic substrate for b-galactosidase. Sometimes you want to know if your mutated gene influences the expression of other genes. influences Sip Mutant cell Prepare cDNA labeled with red or green fluorescent nucleotides WT cell Microarray contains DNA from several thousand different genes, each DNA spotted on a different spot. Red gene expressed in cell1, green gene expressed in cell 2, yellow gene expressed in both cells. A diversity of systems are used for studying development (Model Systems) development Sea Urchins – embryos transparent – see all cells in early embryos. Tunicates – colored cytoplasm – tracing cell lineages. Amphibians – large eggs and embryos – easy to microinject materials into eggs and easy for surgical manipulation. Worms – C. elegans – excellent genetics and can trace the lineage of every cell in the embryo and the adult. Chickens – easy for visualizing. Fruit flys (Drosophila) outstanding for genetics Dictyostelium – easy molecular genetics and outstanding for cell motility. Zebra fish – excellent genetics and a vertebrate Zebra Mice – good genetics but small liters so limits but a vertebrate Mice Basic Mechanisms are Usually Conserved in Different Species Development Provides Insights to Evolution Evolution Now that you have cloned and analyzed the stuck in place gene in a lower eukaryote and shown that it has an important role in cell motility there is a good chance that there should be related gene in a vertebrate genome. A homologous gene homologous Most important developmental genes have homologues in other systems. They will generally control or participate in similar functions. Many times they map to similar gene regions and are clustered with similar genes in both organisms. Often times the differences in their organization and function between lower and higher organisms help us understand how different animal forms evolved. ...
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