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Genomes and Development. An Introduction to Developmental Biology.

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Presentation on theme: "Genomes and Development. An Introduction to Developmental Biology."— Presentation transcript:

1 Genomes and Development

2 An Introduction to Developmental Biology

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

4 The Main Concepts of Developmental Biology: 1)Cell Identity How are cells made different from one another and how do they know what to become? 2)Morphogenesis The creation of form Morph = form, Genesis = create How do cells and tissues take on the proper shapes and architectures? 3)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 How to give daughter cells DIFFERENT Identities Extrinsic MechanismsIntrinsic Mechanisms Localized Cytoplasmic Determinant Secreted Signals Local Cell-cell Interaction

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 Gilbert, 2000 Regulative Development: Twins

12 Gilbert, 2000 Cell-Cell Signaling and Cell Identity 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 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

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

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

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: 1)Cell Identity How are cells made different from one another and how do they know what to become? 2)Morphogenesis The creation of form Morph = form, Genesis = create How do cells and tissues take on the proper shapes and architectures? 3)Differentiation The cell becomes “fully functional” with respect to its role in the tissue to which it belongs

20 Cell IdentityMorphogenesis ? Cell Biology Cell Division/Death Cell Adhesion Cell Movement Cell Shape

21 Morphogenesis Zebrafish Vascular System Weinstein Lab, NIH 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 Fly Tracheal System Human Lung

22 Developmental Biology. S. Gilbert (1)

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

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

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

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

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

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

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

30 Introduction Preparing the Genome Cell Identity Morphogenesis Organogenesis

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

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 1 mm Frog 0.8 mm Fish 0.5 mm Fly 0.05 mm C. elegans 0.15 mm Sea Urchin 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 days90 generations/yr Fly:12 days30 generations/yr Arabidopsis: 6 weeks 8 generations/yr Mouse: 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 HusbandryGreat (cheap and easy)Better (smaller adults, faster maturing) EmbryologyGreat (1 mm)Great (0.7 mm, get more eggs than laevis) Cell BiologySimilar problems with optical clarity for both GeneticsNoneWorking (≈4 month generation time) GenomeAwful (allotetraploid, 3.1 gb) Fine (diploid, 1.7 gb) 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 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

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

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

51 Ceanorhabditis elegans Advantages -Awesome genetics: self-fertilizing hermaphrodite, short generation time -Complete lineage known -Optical clarity -Sequenced Genome -RNAi works particularly well and is systemic Disadvantages -Immunostaining and in situ hybridization challenging -Small embryos -Transgenics not as well developed

52 Drosophila melanogaster Fruit fly Arthropod Invertebrate, Protostome, Ecdysozoan

53 Drosophila melanogaster Advantages -Awesome genetics: short generation time wide array of genetic tools -Excellent cell biology and biochemistry -”Lean” genome Disadvantages -Small embryos -Resistant to transplantation

54 Xenopus laevis African clawed frog Vertebrate Amphibian

55 Xenopus laevis Advantages -Huge embryos: -excellent embryology and biochemistry -rapid “injection assay” for ectopic expression

56 Xenous laevis Embryology Egg diameter: approx. 1 mm

57 Xenopus laevis Advantages -Huge embryos: -excellent embryology and biochemistry -rapid “injection assay” for ectopic expression Disadvantages -Yolky embryo limits optical clarity -No genetics (BUT transgenetics are working in X. laevis and X. topicalis is being developed for “true” genetics)

58 Danio rerio Zebra fish (indigenous to India, but common in pet stores) Vertebrate, Teleost fish

59 Danio rerio Advantages -Best current option for vertebrate forward genetics (based on generation time, space and cost) -Optical clarity and great cell biology

60 Zebrafish embryogenesis

61 Danio rerio Advantages -Best option for vertebrate forward genetics (based on generation time, space and cost) -Optical clarity and great cell biology Disadvantages -Many genetic tools still in development (getting better all the time) -Complex genome? (size and redundancy) zebrafish genome: 1.7 gb pufferfish genome: 0.4 gb

62 Mus musculus Mouse Vertebrate, mammal

63 Mus musculus Advantages -Genetic system that is evolutionarily closest to humans -Good “knockout” and transgenic technology (homologous recombination) -Embryos large enough for dissection and explant assays

64 Mus musculus Advantages -Genetic system that is evolutionarily closest to humans -Good “knockout” and transgenic technology (homologous recombination) -Embryos large enough for dissection and explant assays Disadvantages -In utero development -Limited quantities of embryos -Less practical for genetic screens (although these are in progress in a few places)

65 Other Animal Developmental Models (partial list) Volvox (e.g. Volvox carteri)—colonial algae, models for early multicellular organisms Slime mold (Dictyostelium discoideum)—colonial individual amoebae aggregate to form mobile slug Hydra—cnidarian, “primitive” animal, diploblast (two germ layers w/ no mesoderm) Flatworm (Planaria)—e.g. to study regeneration Leech—study segmentation and neurobiology Sea Urchin (e.g. S. purparatus)—echinoderm, “primitive” deuterostome (evolutionarily closer to humans than Drosophila or C. elegans) Ascidians (tunicate, e.g. Ciona intestinalis)— invertebrate chordates have notochord but no vertebrae, beautiful chordate larvae but “throws it all away” to become sessile, filter-feeding sea squirt Chick (Gallus gallus): Robust embryos, excellent for surgical manipulation Other fish: medaka, puffer fish (Fugu rubpripes, stripped down genome), goldfish Other mammals: rat (larger embryos than mouse), ferret (neurobiology) Plus, “boutique” animals of particular evolutionary importance VolvoxAscidian Chick


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