Developmental Biology Exam I Notes
Developmental Biology Exam I Notes BIOL 3090-001
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This 15 page Class Notes was uploaded by Katherine McKeone on Tuesday September 13, 2016. The Class Notes belongs to BIOL 3090-001 at University of Toledo taught by Robert Steven in Summer 2015. Since its upload, it has received 4 views. For similar materials see Developmental Biology in Biology at University of Toledo.
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Date Created: 09/13/16
Developmental Biology Exam I Stages of Development******** 1. fertilization 2. cleavage 3. gastrulation 4. neurulation: nervous system formation 5. organogenesis: formation of organs 6. metamorphosis: common to frogs and insects, change from larval stage to more complex adult stage. Involves change in morphology of organism “Keep in mind for exam” fucking know it. ******** Embyogenesis Questions of Developmental Biology Differentiation: how does this happen? Morphogenesis: how does this arise? Growth: why does it become larger? Does it deal with complexity? Reproduction: no spontaneous generation of life Evolution (limited time on this section): occurs through changes in developmental processes. Approaches to Developmental Biology anatomical Will reading Baron George Leopold Cuvier’s work help in understanding morphology and anatomical comparisons? experimental genetic Origins of Developmental Biology Aristotle (350BC) preformation: tiny human in sperm Epigenesis: organism is developed from scratch The Cell Theory 18201880 All living organisms consist of cells, the basic unit of life All cells come from other cells The Circle of Life, bitches Somatic vs. Germ Cells distinction made by August Weisman Characteristics of offspring determined by germ cells Somatic cells are cells of the body Germ cells are egg and sperm cells Richard Dawkins Selfish Gene 1 How do Cells Become Different from One Another?**** Mosaic Model: Weisman’s nuclear determinants Wilhelm Roux’s Experiment Half an embryo formed Did not give a whole embryo because the other cell “thought” that the other cell was still there” Does not work in frogs In order for a hypothesis to be “proved” into a theory, does it have to be proved mathmatically? Regulative Development Hans Driesch Regulation occurs Disproved Mosaic Model CellCell Interactions Proven: Induction: cells can induce cells to differentiate Spemann organizer signalling center. Signals to other cells in the area. Resulted in another organism formed New cells tell old cells to grow into a second organism Genetics at the Core of Development Genetics: studying the transmission of inheirited traits Embryology: studying the development of organisms with specific traits 1940s: genes encode proteins Basic Genetics Genotype vs phenotype Genotype: genetic makeup Phenotype: physical expression of genotype allele definition: heterozygous vs homozygous Definition Definition recessive vs dominant definition definition Lecture Two Cleavage division of the fertilized egg into a larger number of cells no increase in cell mass during this stage Different Types of Cleavage***** 2 holoblastic cleavage isolecithal very equal mammals Mesolecithal Zenopus (African clawed frog) some asymmetry Cleaves completely Meroblastic cleavage telolecithal lots of yolk at one end chicken, birds, avian centrolecithal yolk is central just the nuclei divided in one cell Drosophila Does not cleave all the way through Morphogenesis/Pattern Formation Often involves multiple cell behaviors the first pattern formed in development is the overall body plan The Organization of cellular activity in space and time for the formation of structure Morphogenesis: Gastrulation Gastrulation: the dramatic restructuring of the embryo mainly by cell migrations blastula vs gastrula Blastula: cleavage stage Gastrula: movement of cells to generate different layers proteasome vs. deuterostome proteasome: mouth is the first opening to form deuterostome: anus is the first opening to form sea urchins and mammals and vertebrates echinoderms (sea urchins) Morphogenesis: Germ Layers regions of the early embryo that always develop into specific tissue types Triploblasts organisms with three germ layers: endoderm, mesoderm, and ectoderm Endoderm examples Mesoderm layers examples ectoderm layers examples Cell Differentiation The process by which cells become structurally and functionally different from one another gradual 3 at least 250 different cell types in humans Growth very little growth in early development growth occurs in multiple ways increase in cell number increase cell size deposition of extracellular materials Cell Behavior the link between gene action and developmental processes cells then determine the course of development migrations shape changes differential adhesion division cellcell signaling cell death Gene Expression and Protein Synthesis Genes are passive participants in development determine when and where proteins are produced Multiple ways to control protein synthesis not really interested in “housekeeping genes” Complex Control Regions Regulate Gene Expression differentiation: generated by differences in gene expression transcription factors or gene regulatory proteins binds to cisregulatory modules modules can be interdependent Development is Progressive ***** complexity of the embryo gradually increases over time fate, determination, and specification Fate: final outcome, expected result of normal development Determination: stable change in the internal state of a cell, such that the cell’s state is now fixed. Specification: when isolated and cultured in the neutral environment of a simple culture medium away from the embryo, they develop according to their normal fate. Inductive Interactions a signal from one cell or tissue influences the development of an adjacent group of cells can make cells different from one another distance of action is variable permissive vs. instructive permissive is a choice of two things Signal Transduction transmission of the signal into the interior of the cell relays of proteinprotein interactions within the cell 4 phosphorylation is key Cells must be competent to Receive a Signal example is lens induction small distances Genetic Specificity of Induction Patterning from Positional Information***** Cells can acquire an identity based on their position Once positional value is obtained cells interpret it to differentiate properly Position signaled by a morphogen: A chemical whose concentration varies and which is involved in pattern formation A substance involved in pattern formation whose spatial concentration varies and to which cells respond differently at different threshold concentrations Cytoplasmic Determinants and Asymmetric Divisions Certain molecules (determinants) are distributed unevenly within the cell Determinants distributed unevenly during cell division Determinants at some point specify a particular fate to the cell that contains them Asymmetric Divisions: Stem Cells Stem cells undergo a special type of asymmetric cell division Undifferentiated cells that divide to produce more stem cells along with other cells that go n to differentiate Capable of repeated divisions Pluripotent Give many different types of cell types Hematopoietic stem cells Totipotent Go on to give a whole organism Zygote DNA is NOT a Blueprint for an Organism DNA does not contain a full description of the organism to which it will give rise The genome instead contains a generative program of instructions for making the organism DNA is a blueprint for the primary structure of proteins Lecture Three Drosophila as a Model Organism Originally for studies on the rules of genetic inheritance Small (3mm), easy to handle, short (2 weeks) life cycle Used in genetic screens for development genes Similar genes are found in mammals Drosophila Embryo Cleavage Rapid nuclear division, without the formation of cells Creates syncytium: cell with multiple nuclei and a common cytoplasm 5 After three hours membranes grow in from the surface to enclose nuclei and form cells Drosophila Gastrulation Mesoderm (muscle); ventral Endoderm (gut); poles Both move inside during gastrulation Mesoderm moves first forming a ventral furrow; then a tube Ectoderm remains as the outer layer forming an epidermis Inward movement of cells Migrating mesodermal cells create the ventral flow Segmentation becomes visible during germband extension Drosophila First Instar Larva Acron: anterior specialized structure Telson: posterior specialized structure Denticles: small toothlike outgrowths from the surface of the larvae Each bristle segment has a recognizable denticle and bristle pattern CLICKER: Drosophila embryos are classified as centrolecithal Patterning of the Drosophila Embryo AP and DV axes laid out in egg AP axis in embryo are divided into parasegments Individual developmental units (compartments) that give rise to the segments Screening for Developmental Mutants***** Screen EMS mutagen Balancer chromosome Saturating screen When screen is done enough, you start finding the same type of mutations Maternal Effect Mutations The genotype of the mother is expressed in the phenotype of its offspring Paternal contribution has no effect Setting Up the Body Plan: Sequential Gene Expression***** Maternal genes: expressed by the mother in the tissues of the ovary Maternal gene products laid down in egg They provide positional information This info activates the zygotic genes, which act sequentially. Specification of the AP Axis Roles of maternal genes identified by the effects of mutations Three classes of maternal genes anterior posterior Terminal CLICKER A protein that is found in a concentration gradient and influences a cell’s developmental outcome is called a morphogen 6 Bicoid is Localized in the Anterior mRNA distribution is analyzed by in situ hybridization Protein distribution is determined by antibody staining After fertilization, Bicoid protein is produced in the anterior but diffuses to produce AP gradient. Bicoid is a Morphogen Conclusion based on genetic and physical experiments Cytoplasm transfer experiments reveal the importance of Bicoid A transcription factor Controls GAP genes Lecture Four Screening for Developmental Mutants Screen EMS mutagen Balancer chromosome Saturating screen Posterior Group Maternal Genes Gradients of nanos and caudal control posterior patterning Caudal is a morphogen Also specify the germplasm in the egg Nanos Protein Functions to Establish a Maternal Gradient of Hunchback Protein Nanos is not a morphogen Nanos suppresses the translation of maternal hunchback mRNA. Loss of Maternal hunchback Suppresses nanos Mutants Remove the maternal hunchback protein in nanos mutant animals These animals have a WT phenotype Conclusion: Nanos functions to decrease maternal hunchback in the embyro Caudal Also required to pattern the posterior embryo Caudal mutants have missing abdominal segments Maternal mRNA is evenly distributed Posterior to anterior protein gradient formed by inhibition of Caudal protein synthesis by Bicoid Green bicoid, red caudal Terminal Group Maternal Genes Specify structures at the ends of the embryo Terminal regions are specified together (same molecular pathway), despite the distance between them Torso is a Receptor Tyrosine Kinase Distributed everywhere in the membrane of the fertilized egg Activated by a ligand only present at the ends of the egg 7 Ligand is the trunk protein Signal transduction pathway activates zygotic genes in the termini DorsalVentral Polarity Ligand: Spatzle fragment Expressed in ventral follicle cells in the ovary Receptor: Toll Present throughout the membrane of the egg Activated Toll causes the transcription factor Dorsal to enter the nearby nuclei Toll Activation Affects Dorsal Localization Dorsal is a morphogen The Toll Signaling Pathway***** Activated Toll binds to an adaptor protein This initiates a pathway leading to the breakdown of the inhibitory protein Cactus Dorsal is free to move into the nucleus NFkB in mammals Toll pathway is multifunction CLICKER EMBRYOS WITH DEFECTIVE DORSAL GENE ARE DORSALIZED Drosophila Egg Development Germline stem cell divides to generate a stem cell and a cytoblast, which divides to give 16 cells One becomes the oocyte Others are nurse cells Follicle cells have a critical role in patterning Cells of the mother Somatic cells A/P Polarity: Signals from Older to Younger Egg Chambers Polarize the Oocyte First evidence of A/P polarity is the posterior positioning of the oocyte in the cyst. A series of signals from the previous cyst determines which germ cell will develop into the oocyte Oocyte and posterior follicle cells specifically express the adhesion molecule E cadherin. OocyteFollicle Cell Interactions A/P and D/V axes are established by interactions involving Gurken Activates the receptor Torpedo Torpedo is a RTK Signal specifies posterior follicle cells Follicle cells signal back causing rearrangement of cytoskeleton Gurken further induces Dorsal follicle cells. mRNA Localization in the Egg Bicoid mRNA transported to anterior on MT (microtubules) towards the “” end Nanos mRNA transported to posterior on MT towards “+” end 8 CLICKER Follicle cells are part of the ovary Clicker A saturating screen means multiple alleles were found for each gene identified in the screen. Dorsal Protein Controls the Expression of Zygotic Genes on the DV Axis Dorsal activity defines broad regions of the DV axis Has two types of activities: activation or repression of genes Twist and snail are activated by high levels of Dorsal (ventral) Rhomboid activated by low levels of Dorsal Dpp repressed by Dorsal Concentration of Dorsal Determines Which Genes are Activated Decapentaplegic is a Morphogen for the Dorsal Region Embryo now cellular; transcription can’t diffuse between nuclei Dpp is a secreted signalling protein (TGFbeta family member) Lecture Five The Effects of Mutations in the Zygotic Developmental Genes Gap mutants lack large regions of the body Activates Pair Rule Pair rule mutants lack portions of every other segment Activates segmentation Segmentation mutants have defects in every segment. The AP Axis is Divided into Broad Regions by Gap Gene Expression Gap genes are the first zygotic genes expressed along the AP Axis All encode transcription factors Bicoid activates hunchback, which activates other Gap genes mutants : large regions of body pattern missing in the AP axis Missing regions, in general, correspond to where the gene is expressed. Maternal Bicoid Protein Controls Zygotic hunchback Expression Hunchback is turned on only when the transcription factor Bicoid is at a certain threshold level This level obtained only in the anterior Bicoid binds the hunchback Promoter Hunchback expression can be monitored using a lacZ reporter fusion gene Hunchback promoter fused to the bacterial lacZ gene Blue stain represents the level of expression Promoter “bashing” experiments defined an essential 263 bp region Hunchback Protein Activates and Represses Other Gap Genes Hunchback is another transcription factor that acts as a morphogen Limits of gap gene expression determined by Bicoid and Hunchback Example: kruppel activated by Bicoid and intermediate levels of hunchback 9 High Hunchback levels represses kruppel expression. Relationship Between Parasegments and Segments Segmentation an obvious feature of larvae Parasegments: Individual developmental units (compartments) in the Drosophila embryo that gives rise to segments Visualized by gene activity Offset from segments by half a segment Expression of PairRule Genes Genes are expressed in 7 stripes just before cellularization All pairrule genes expressed in alternate parasegments Generation of the PairRule Stripes Bicoid and Hunchback activate eve in a broad domain Giant and Kruppel proteins represses eve to define the borders Repressors override activators One evenskipped cisControl Region The promoter region is complex Seven different ciscontrol regions: one for each stripe Each region has multiple binding sites for each of the transcription factors CLICKER Are all transcription factors morphogens? No Segmentation Genes Activated in response to the pairrule genes Expressed in 14 transverse stripes Example is engrailed Engrailed Delimits the Anterior Parasegment Boundary Expressed throughout the life of the fly Delimits a celllineage boundary Defines a compartment Segmentation Genes Affect segmentations patterns visualized on the larval cuticle Normal patterning depends on the stabilization of segment boundaries Wg and hh mutants have denticles completely covering abdomen Anterior region duplicated in mirror image Expression of Segmentation Genes Segmentation genes stabilize segment boundaries Hh and wg expressed on either side of the parasegment boundary Patched localized to cells without hh. The Hedgehog and Wingless Signalling Pathways Pathways involved in the intracellular signalling circuit set up between adjacent cells, which delimits the bundary between them Hh and Wg are the secreted signal proteins (ligands) 10 Patched and Frizzled are receptors Aslo involved in other aspects of development Important roles in vertebrates as well Hedgehog signaling Pathway Wingless Signalling Pathwya Dropsophila Hox genes are controlled by pair rule genes Gastrulation occurs mainly because of this behavior: cell migrations Wg and Hh Signaling Circuit En expressing cells secret Hh Hh diffues over the compartment boundary to activate and maintain wg expression in the adjacent cells Wg feeds back to maintain hh and en expression in cell’s posterior Segment Identity is Specified by Hox Gene First evidence of genes that specify segment identity came from the observation of mutations that produced homeotic transformations Genes affected are called Hox genes These contain a homeobox Considered one of the fundamental defining features of multicellular animals All act in a similar way, specifying identity along the A/P axis Drosophila HOM Complexes Two Hox gene clusters in Drosophila Antennapedia complex controls the development of anterior parasegments Bithorax complex controls posterior parasegments Striking colinearity between the spatial and temporal gene expression patterns and their order on the chromosome Bithorax Complex Mutations Flies with bithoraxoid mutation have the anterior portion of the haltere transformed into part of a wing A double mutant of bithoraxoid and postbithorax results in a complete wing instead of a haltere. Antennapedia Complex Mutations Flies with the dominant Antennapedia mutation have… Combinatorial Activity of the Hox Gene Defines the character of each segment Gap and pairrule genes determine the bithorax gene expression pattern Conclusion: segment identities are likely determined by variations in the spatial and temporal expression patterns of the Hox gene. Other Points about the Hox Gene Many transcription factors contain homeobox sequences, but are not part of the Hox gene clusters 11 We don’t know the downstream targets of the Hox genes. Lecture Six Vertebrate Body Plan Vertebral column: segmented backbone that surrounds the spinal cord Defines the A/P axis D/V axis: mouth ventral, spine dorsal Mainly bilaterally symmetric Differences and Similarities Egg size Different appearance before gastrulation Different amount of yolk Phylotypic stage: stage after gastrulation when embryos appear similar Xenopus laevis African clawfrog Develops normally in tap water Eggs are easy to obtain Eggs are large and hardy enough for manipulation Fragments and cultures well Xenopus egg Distinct polarity Animal region: dark and pigmented Vegetal region: pale, yolky, and heavy Cleavage of the Xenopus Embryo No cell growth so cells become smaller with each division Cleavage occurs synchronously Blastomeres: Cells derived from cleavage divisions Blastula: Spherical mass of cells with a fluid filled cavity inside; cavity is called the blastocoel Xenopus Gastrulation***** Gastrulation begins at the blastopore Involution Epiboly: the spreading of the ectoderm to cover the whole embryo Archenteron Amphibian Neurulation First indication is formation of neural folds Embryo is called a nerula This tissue folds and fuses to form the neural tube Neural crest cells detach from the tube to form other tissues Chicken Life Cycle Similar development to mammals especially in gastrulation and later Easier to obtain and visualize Chicken Egg 12 Blastoderm: flat embryo several cells thick Albumen: egg white Chalaza: cord like balancer for the yolk Shell is made of calcium carbonate Cleavge and Epiblast Formation Hypoblast: layer of cells forming the floor of the cavity Epiblast: remaining blastoderm that forms the embryo Gastrulation begins with the formation of the primitive streak Ingression: Cells move inward individually (not as a sheet) Mesenchyme: a loose collection of cells Chick Gastrulation Hensen’s node: a condensation of cells at the anterior end of the primitive streak in chick and mouse embryos. Regression of Hensen’s Node: After inward cell migrations stop the primitive streak regresses Notochord forms from mesoderm anterior to the regressing node Neurulation in the Chick Embryo Neural folds on either side of the neural plate Progression anterior to posterior Discussion Questions from Blackboard Gain of function mutations are typically dominant in nature while loss of function are recessive. Chick Embryo Development Somite: areas of development 13, 22, and 40 somite stages Stained with the early mesoderm and notochord marker Brachyury ExtraEmbryonic Structures of the Chick Embryo***** Membranes: Chorion, allantois, amnion, and yolk sac Fluidfilled amniotic cavity provides protection Allantosis: site of oxygen and carbon dioxide exchange Lecture Seven CLICKER QUESTION Do the embyros at the phylotypic stage look similar in all species? Yes Mouse Life cycle 9 week life cycle is relatively short for mammals Egg is small and has no yolk Cleavage in the Mouse Embryo***** Asynchronous cell divisions: Every 12 hours (relatively slow) 13 After the eight cell stage, compaction occurs to form the morula The early cleavage stage embryo (a solid ball of cells) Blastocyst: Composed of inner cell mass and trophectoderm ICM forms the embryo and extraembryonic structures Trophectoderm forms just extraembryonic structures Ex: placenta Came from the embryo but do not form the final embryo product. First Differentiation Event Early mouse embryo is highly regulative; cells determined only after 32 cell stage Argues against the importanc of maternal determinants All cells are totipotent up until the 32cell stage. After this point, the cells can only become cells of the embryo and are therefore pluripotent. Regulation in Mouse Embryo Development Can pull cells out to generate cell lines Can take cells out and embryo will be fine, add them and the embryo will be fine This indicates that there is regulation involved to form the appropriately sized mouse. PostImplantation Development ICM divides into two regions: epiblast and primitive endoderm Primitive endoderm contributes to extraembryonic membranes Epiblast develops into the embryo (and some extraembryonic structures) Pluripotent Cup shape is peculiarity of rodents: other mammals (vertebrates??) like chick and humans have flat early embryos. Gastrulation in the Mouse Embryo Epiblast cells converge in the posterir and migrate through to spread out laterally and anteriorly Node: Equivalent to Spemann organizer region Clicker Which region gives rise to all embryonic tissues? Epiblast Human Life Cycle 38 weeks from fertilization to birth Embro: between implantation and 8 weeks Fetus: after 8 weeks until birth Early Mammalian Development Placenta: structue formed in th euterine wall where the blood systems of mother and embryo interfce to exchange nutrients and waste products. Ectopic pregnancy: when the egg implants in the fallopian tube Human Embryos are Regulative: Monozygotic Twins Indentical twins Have the same set of DNA 14 Embryo splits early in developments. Can have two chorians Can have one chorian but two amnions Can have one amnion (can result in conjoined twins) Human Gastrulation and Changes in the Extraembryonic Membranes Amniocentesis: The sampling and analysis of fluid from the amniotic cavity, usually performed at 1420 weeks Checking the genetics of that embryo Clicker The cells of the ICM are pluripotent Sperm Entry Defines the Future Dorsal side of the Xenopus Embryo***** Sperm entry occurs anywhere in the animal hemisphere In regards to sperm entry of an amphibian: Could there be a cell surface receptor on the particular hemisphere? Cortical rotation: cytoplasm in the cortex loosesn and rotates away from the site of sperm entry Involves the orientation of MTs away from the sperm entry site Maternal factors: beomces relocated opposite site of sperm entry. Wnt Signaling Pathway Segmentation gene Activates things on the dorsal side of the embryo Clicker The position of the Xenopus blastopore is determined by the site of sperm entry anf Wnt signaling Beta Catenin Has a Dorsalizing Effect Injection of betacatenin results… Gravity Defines the Chick A/P Axis 15
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