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Genomes and Development

Genomes and Development. An Introduction to Developmental Biology. Mouse. Sea Urchin. Xenopus. An Introduction to Developmental Biology. Ascidian. Zebrafish. Drosophila. C. elegans. The Main Concepts of Developmental Biology: Cell Identity

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Genomes and Development

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  1. Genomes and Development

  2. An Introduction to Developmental Biology

  3. Mouse Sea Urchin Xenopus An Introduction to Developmental Biology Ascidian Zebrafish Drosophila C. elegans

  4. The Main Concepts of Developmental Biology: Cell Identity How are cells made different from one another and how do they know what to become? Morphogenesis The creation of form Morph = form, Genesis = create How do cells and tissues take on the proper shapes and architectures? Differentiation The cell becomes “fully functional” with respect to its role in the tissue to which it belongs

  5. Cell Identity (Cell Fate Specification) 1) Identity is a continuum: Naïve--specified--determined--differentiated Reversible vs. Irreversible (stable epigenetic state) Cell transplantation can distinguish between reversible and irreversible cell fate

  6. Cell Fate Specification is a PATH, not a Binary Decision

  7. Cells are reprogrammed according to new environment Cells retain original identity

  8. Cell Identity (Cell Fate Specification) 1) Identity is a continuum: Naïve--specified--determined--differentiated Reversible vs. Irreversible Cell transplantation can distinguish between reversible and irreversible cell fate • 2) Mechanisms for specification • Intrinsic vs. Extrinsic

  9. Intrinsic Mechanisms How to give daughter cells DIFFERENT Identities Extrinsic Mechanisms Local Cell-cell Interaction Localized Cytoplasmic Determinant Secreted Signals

  10. Cell Identity (Cell Fate Specification) 1) Identity is a continuum: Naïve--specified--determined--differentiated Reversible---Irreversible Cell transplantation can distinguish between reversible and irreversible cell fate • 2) Mechanisms for specification • Intrinsic • Cell autonomous (e.g.: Localized cytoplasmic determinants) • Independent of environment • Mosaic Development: “patchwork” that is difficult to repair if part is damaged or lost Extrinsic Cell non-autonomous Cell identity is dependent on environment (condition) E.g. Extracellular signals that control cell identity Regulative Development: if some parts are lost, others may be able to respond to signals in their place

  11. Regulative Development: Twins Gilbert, 2000

  12. Cell-Cell Signaling and Cell Identity A small number of signaling pathways control all cell-cell communication What provides the specificity? Context: Other signals received at the same time History: A cell’s current identity influences how it responds to new signals Tyrosine kinase receptors (EGF-R, FGF-R, etc.) hedgehog wingless (wnt) TGF-ß/BMP/Activin Notch Toll Jak/Stat Toll/IL Tor G Protein Coupled Receptors Nuclear Hormone Receptors Gilbert, 2000

  13. Morphogen: a factor that controls cell identity by acting at a distance and in a concentration-dependent manner (different concentrations= different identities) (Lewis Wolpert, 1969)

  14. What is Cell Identity?

  15. All cells contain the same genome (mostly) Somatic cell nuclear transfer (cloning) Nuclei from differentiated somatic cells can give rise to complete, fertile adult when activated by egg cytoplasm Frogs: Gurdon and Uehlinger, 1966 Sheep (Dolly): Wilmut et al., 1997 Since cells with different identities contain the same genomes, CELL IDENTITY = DIFFERENTIAL USAGE OF THE GENOME

  16. What is Cell Identity? Cell Identity = Differential Utilization of the Genome Cell Identity = Specific Pattern of Gene Expresson

  17. Mother Nature Controls Gene “Expression” at EVERY Level DNA Transcription Alternative Splicing RNA Stability RNA Localization RNA Translation Protein Stability Protein Modification Protein Localization Protein-Protein Interaction Protein

  18. What is Cell Identity? Cell Identity = Differential Utilization of the Genome Cell Identity = Specific Pattern of Gene Expresson and Genes that can be Expressed

  19. The Main Concepts of Developmental Biology: Cell Identity How are cells made different from one another and how do they know what to become? Morphogenesis The creation of form Morph = form, Genesis = create How do cells and tissues take on the proper shapes and architectures? Differentiation The cell becomes “fully functional” with respect to its role in the tissue to which it belongs

  20. ? Cell Biology Cell Division/Death Cell Adhesion Cell Movement Cell Shape Cell Identity Morphogenesis

  21. Morphogenesis • Factors Affecting Morphogenesis • 1) Cell number (Cell division and cell death) • 2) Cell Shape • 3) Cell-Cell Affinity (Adhesion) • 4) Cell Polarity • 5) Cell Movement (Migration) • 6) Coordinated Growth Human Lung Fly Tracheal System Zebrafish Vascular System Weinstein Lab, NIH

  22. (1) Developmental Biology. S. Gilbert

  23. (2) (1) Developmental Biology. S. Gilbert

  24. (3) Axis Specification (2) (1) Developmental Biology. S. Gilbert

  25. (3) Axis Specification (2) (1) (4) Developmental Biology. S. Gilbert

  26. (3) Axis Specification (2) (1) (4) (5) Developmental Biology. S. Gilbert

  27. (3) Axis Specification (2) (1) (4) (5) (6) Metamorphosis Developmental Biology. S. Gilbert

  28. (3) Axis Specification (2) (1) (7) (4) (5) (6) Metamorphosis Developmental Biology. S. Gilbert

  29. (3) Axis Specification (8) Aging and Death (2) (1) (7) (4) (5) (6) Metamorphosis Developmental Biology. S. Gilbert

  30. Introduction Preparing the Genome Cell Identity Morphogenesis Organogenesis

  31. Mouse Ascidian (sea squirt) Xenopus How to choose a model systemOr, Why do Developmental Biologists study these bizarre creatures? Zebrafish Chicken Drosophila C. elegans

  32. Some questions Developmental Biologists ask: Where do these cells come from and what do they do? Fate mapping and lineage analysis -Injection/activation of lineage tracer -Genetic lineage analysis Cell transplantation What genes are important for the developmental process I am studying? -Genetic screens/genetic mapping -Expression profiling Where is the gene I am studying expressed? -In situ hybridization -Expression profiling -Immunofluorescence -In vivo imaging What is the function of the gene I am studying and where does it act? -Loss of function by RNAi and morpholino -Targeted gene knockouts -Mis-expression -Mosaic analysis

  33. How to choose a model system -Different animal species offer different experimental advantages -Comparative studies provide a more complete understanding -Strong evolutionary conservation of developmental mechanisms

  34. How to choose a model system 1) Animal husbandry -Want large numbers of embryos -Want to control timing (i.e. fertilization) -Early work done on marine organisms e.g. Marine Biological Laboratory, Woods Hole, MA -Best if not limited to mating seasons -Most current work done on animals raised in lab

  35. How to choose a model system 2) Embryology -Many developmental biology experiments involve physically manipulating embryo -moving or altering division of early blastomeres (cells) -dissection and reconstitution -cell or tissue transplantation -injection of DNA, RNA or cell lineage markers -Bigger is often better for these experiments -Some embryos are more robust than others -External development or in vitro culturing is important (can do some injections in utero and some embryo culturing in vitro)

  36. 0.5 mm Fly 0.15 mm Sea Urchin 1 mm Frog 0.05 mm C. elegans 0.8 mm Fish Embryos are to scale

  37. How to choose a model system 3) Cell Biology and Microscopy -Need to deal with protective layers (egg shell, vitelline envelope) -Ease of fixation and staining (e.g. immunostaining or in situ hybridization) -Tissue thickness -Optical clarity -In vivo imaging (clarity, ability to express transgenes)

  38. PAR2-GFP

  39. How to choose a model system 4) Biochemistry -Material limiting: need to be able to harvest large amounts of embryos -Extracts need to “behave well” (stable proteins, ease of fractionation)

  40. How to choose a model system 5) Genetics -Need to grow for many generations or indefinitely in lab -Generation time is limiting: the shorter the better Worm: 4 days 90 generations/yr Fly: 12 days 30 generations/yr Arabidopsis: 6 weeks 8 generations/yr Mouse: 10-11 weeks 5 generations/yr Zebrafish: 3 months 4 generations/yr -Forward genetics (mutational analysis)--need to keep a large number of families in a small space -Reverse genetics—ability to “knock out” a given gene of interest -Transgenetics—ability to put back new or modified genes into genome

  41. How to choose a model system 6) Genomics -Genome sequence available -Low genome complexity (less “junk” DNA and smaller regulatory regions) -Low amount of gene redundancy makes forward and reverse genetics easier

  42. Genome Comparison

  43. Pufferfish Tedraodon nigroviridis Genome: 385 Mbp Zebrafish Danio rerio Genome: 1,933 Mbp African lungfish Protopterus aethiopicus Genome: 130,000 Mbp

  44. Fruitfly D. melanogaster Genome: 170 Mbp Silk moth Bombyx mori Genome: 530 Mbp Honeybee Apis mellifera Genome: 1,770 Mbp

  45. How to choose a model system 6) Genomics -Genome sequence available -Low genome complexity (less “junk” DNA and smaller regulatory regions) -Low amount of gene redundancy makes forward and reverse genetics easier -Organism’s place on evolutionary tree

  46. Animal Evolutionary Tree Snails Planaria Hydra Sponges

  47. How to choose a model system 6) Genomics -Genome sequence available -Low genome complexity (less “junk” DNA and smaller regulatory regions) -Low amount of gene redundancy makes forward and reverse genetics easier -Organism’s place on evolutionary tree -Comparative Genomics

  48. Our current model systems were chosen for historical reasons Case study: Xenopus laevis vs. Xenopus tropicalis CharacteristicX. laevisX. tropicalis Husbandry Great (cheap and easy) Better (smaller adults, faster maturing) Embryology Great (1 mm) Great (0.7 mm, get more eggs than laevis) Cell Biology Similar problems with optical clarity for both Genetics None Working (≈4 month generation time) Genome Awful (allotetraploid, 3.1 gb) Fine (diploid, 1.7 gb) http://faculty.virginia.edu/xtropicalis/ Genome differences b/w laevis and tropicalis known for 30 years, why didn’t people switch? -Genetics was only beginning to be applied to development -Genomics as a useful tool was not even on the horizon Factors affecting why certain model systems become “entrenched”: Historical inertia: community of researchers all trained in a particular system Technical inertia: accumulated tools and resources for one system cannot be transferred--can lose decades of experimental time when switching

  49. Ceanorhabditis elegans Soil-dwelling roundworm Phylum Nematoda--Nematodes Invertebrate, Protostome, Ecdysozoan Adult= approx 1 mm long Movie credits: Goldstein Lab, UNC

  50. Ceanorhabditis elegans Advantages -Awesome genetics: self-fertilizing hermaphrodite, short generation time -Complete lineage known -Optical clarity

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