Intro.lecture.2012

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Genomes and Development
An Introduction to
Developmental Biology
Mouse
Sea Urchin
Xenopus
An Introduction to
Developmental Biology
Drosophila
Zebrafish
Ascidian
C. elegans
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
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
Cell Fate Specification is a PATH, not a Binary Decision
Cells are reprogrammed
according to new
environment
Cells retain original
identity
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
How to give daughter cells DIFFERENT Identities
Intrinsic Mechanisms
Localized Cytoplasmic
Determinant
Extrinsic Mechanisms
Secreted Signals
Local Cell-cell
Interaction
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
Regulative Development: Twins
Gilbert, 2000
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
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)
What is Cell Identity?
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
What is Cell Identity?
Cell Identity = Differential Utilization of the Genome
Cell Identity = Specific Pattern of Gene Expresson
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
What is Cell Identity?
Cell Identity = Differential Utilization of the Genome
Cell Identity = Specific Pattern of Gene Expresson
and Genes that can be Expressed
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
Cell Identity
?
Morphogenesis
Cell Biology
Cell Division/Death
Cell Adhesion
Cell Movement
Cell Shape
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
(1)
Developmental Biology. S. Gilbert
(1)
Developmental Biology. S. Gilbert
(2)
(3) Axis Specification
(1)
Developmental Biology. S. Gilbert
(2)
(3) Axis Specification
(1)
(2)
(4)
Developmental Biology. S. Gilbert
(3) Axis Specification
(1)
(2)
(4)
(5)
Developmental Biology. S. Gilbert
(3) Axis Specification
(1)
(2)
(4)
(5)
(6) Metamorphosis
Developmental Biology. S. Gilbert
(3) Axis Specification
(1)
(2)
(7)
(4)
(5)
(6) Metamorphosis
Developmental Biology. S. Gilbert
(3) Axis Specification
(8) Aging
and Death
(1)
(2)
(7)
(4)
(5)
(6) Metamorphosis
Developmental Biology. S. Gilbert
Introduction
Preparing the Genome
Cell Identity
Morphogenesis
Organogenesis
Mouse
Ascidian (sea squirt)
Xenopus
How to choose a model system
Or, Why do Developmental Biologists study these bizarre creatures?
Drosophila
Zebrafish
Chicken
C. elegans
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
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
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
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)
0.5 mm
Fly
1 mm
Frog
0.15 mm
Sea Urchin
0.05 mm
C. elegans
0.8 mm
Fish
Embryos are to scale
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)
PAR2-GFP
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)
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
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
Genome Comparison
Pufferfish
Tedraodon nigroviridis
Genome: 385 Mbp
Zebrafish
Danio rerio
Genome: 1,933 Mbp
African lungfish
Protopterus aethiopicus
Genome: 130,000 Mbp
Fruitfly
D. melanogaster
Genome: 170 Mbp
Silk moth
Bombyx mori
Genome: 530 Mbp
Honeybee
Apis mellifera
Genome: 1,770 Mbp
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
Animal Evolutionary Tree
Snails
Planaria
Hydra
Sponges
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
Our current model systems were chosen for historical reasons
Case study: Xenopus laevis vs. Xenopus tropicalis
Characteristic
X. laevis
X. 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
Ceanorhabditis elegans
Soil-dwelling roundworm
Phylum Nematoda--Nematodes
Invertebrate, Protostome, Ecdysozoan
Adult= approx 1 mm long
Movie credits: Goldstein Lab, UNC
Ceanorhabditis elegans
Advantages
-Awesome genetics: self-fertilizing hermaphrodite, short
generation time
-Complete lineage known
-Optical clarity
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
Drosophila melanogaster
Fruit fly
Arthropod
Invertebrate, Protostome, Ecdysozoan
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
Xenopus laevis
African clawed frog
Vertebrate Amphibian
Xenopus laevis
Advantages
-Huge embryos:
-excellent embryology and biochemistry
-rapid “injection assay” for ectopic expression
Xenous laevis Embryology
Egg diameter: approx. 1 mm
Advantages
Xenopus laevis
-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)
Danio rerio
Zebra fish (indigenous to India, but common in pet stores)
Vertebrate, Teleost fish
Danio rerio
Advantages
-Best current option for vertebrate forward genetics
(based on generation time, space and cost)
-Optical clarity and great cell biology
Zebrafish embryogenesis
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
Mus musculus
Mouse
Vertebrate, mammal
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
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)
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
Volvox
Ascidian
Chick

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