Genomes and Development. An Introduction to Developmental Biology.

Genomes and Development

An Introduction to Developmental Biology

An Introduction to Developmental Biology

Mouse

DrosophilaZebrafish

Xenopus

Ascidian

C. elegans

Sea Urchin

The Main Concepts of Developmental Biology:

1) Cell IdentityHow are cells made different from one another and how do theyknow what to become?

2) MorphogenesisThe creation of form Morph = form, Genesis = createHow do cells and tissues take on the proper shapes and

architectures?

3) DifferentiationThe cell becomes “fully functional” with respect to its role in thetissue 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


1) Identity is a continuum:Naïve--specified--determined--differentiated Reversible vs. IrreversibleCell transplantation can distinguish between reversible and


2) Mechanisms for specificationIntrinsic vs. Extrinsic

How to give daughter cells DIFFERENT Identities

Extrinsic MechanismsIntrinsic Mechanisms

Localized CytoplasmicDeterminant

Secreted Signals Local Cell-cellInteraction


1) Identity is a continuum:Naïve--specified--determined--differentiated Reversible---IrreversibleCell transplantation can distinguish between reversible and


2) Mechanisms for specificationIntrinsicCell autonomous (e.g.: Localized cytoplasmic determinants)Independent of environmentMosaic Development: “patchwork” that is difficult to repair if part is

damaged or lost

ExtrinsicCell non-autonomousCell identity is dependent on environment (condition)

E.g. Extracellular signals that control cell identityRegulative Development: if some parts are lost, others may be able

to respond to signals in their place

Gilbert, 2000

Regulative Development: Twins

Gilbert, 2000

Cell-Cell Signaling and Cell Identity

Tyrosine kinase receptors (EGF-R, FGF-R, etc.)hedgehogwingless (wnt)TGF-ß/BMP/ActivinNotchTollJak/StatToll/ILTorG Protein Coupled ReceptorsNuclear Hormone Receptors

A small number of signaling pathways control all cell-cell communication

What provides the specificity?Context: Other signals received at the same timeHistory: A cell’s current identity influences how it responds to new signals

(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)

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, 1966Sheep (Dolly): Wilmut et al., 1997

Since cells with different identities contain the same genomes,

CELL IDENTITY = DIFFERENTIAL USAGE OF THE GENOME


Cell Identity = Differential Utilization of the Genome

Cell Identity = Specific Pattern of Gene Expresson

RNA

DNA

Protein

TranscriptionAlternative SplicingRNA StabilityRNA Localization

TranslationProtein StabilityProtein ModificationProtein LocalizationProtein-Protein Interaction

Mother Nature Controls Gene “Expression” at EVERY Level


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 IdentityHow are cells made different from one another and how do theyknow what to become?

2) MorphogenesisThe creation of form Morph = form, Genesis = createHow do cells and tissues take on the proper shapes and

architectures?

3) DifferentiationThe cell becomes “fully functional” with respect to its role in thetissue to which it belongs

Cell Identity Morphogenesis?

Cell BiologyCell Division/Death Cell Adhesion Cell Movement Cell Shape

Morphogenesis

Zebrafish Vascular System Weinstein Lab, NIH

Factors Affecting Morphogenesis1) Cell number (Cell division and cell

death)2) Cell Shape3) Cell-Cell Affinity (Adhesion)4) Cell Polarity5) Cell Movement (Migration)6) Coordinated Growth

Fly Tracheal System

Human Lung

Developmental Biology. S. Gilbert

(1)


(1) (2)


(1) (2)

(3) Axis Specification


(1) (2)


(4)


(1) (2)


(4)

(5)


(1) (2)


(4)

(5)

(6) Metamorphosis


(1) (2)


(4)

(5)

(6) Metamorphosis

(7)


(1) (2)


(4)

(5)

(6) Metamorphosis

(7)

(8) Aging and Death

Introduction

Preparing the Genome

Cell Identity

Morphogenesis

Organogenesis

Mouse

DrosophilaZebrafish

Ascidian (sea squirt)Xenopus

Chicken

C. elegans

How to choose a model systemOr, Why do Developmental Biologists study these bizarre

creatures?

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


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)

1 mmFrog

0.8 mmFish

0.5 mm Fly

0.05 mm C. elegans

0.15 mmSea Urchin

Embryos are to scale


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


4) Biochemistry-Material limiting: need to be able to harvest large

amounts of embryos

-Extracts need to “behave well” (stable proteins, ease of fractionation)


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


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

PufferfishTedraodon nigroviridis

Genome: 385 Mbp

ZebrafishDanio rerio

Genome: 1,933 Mbp

African lungfishProtopterus aethiopicusGenome: 130,000 Mbp

FruitflyD. melanogaster

Genome: 170 Mbp

Silk mothBombyx mori

Genome: 530 Mbp

HoneybeeApis mellifera

Genome: 1,770 Mbp





-Organism’s place on evolutionary tree

Animal Evolutionary Tree

Snails

Planaria

Hydra

Sponges





-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/

Factors affecting why certain model systems become “entrenched”:Historical inertia: community of researchers all trained in a particular systemTechnical 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

Ceanorhabditis elegans

Adult= approx 1 mm long

Soil-dwelling roundworm

Phylum Nematoda--Nematodes

Invertebrate, Protostome, Ecdysozoan

Movie credits: Goldstein Lab, UNC

Ceanorhabditis elegans

Advantages-Awesome genetics: self-fertilizing hermaphrodite, short generation time

-Complete lineage known

-Optical clarity

Ceanorhabditis elegansAdvantages-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 melanogasterFruit 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 laevisAfrican 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

Xenopus laevisAdvantages-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 rerioZebra 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 musculusMouse

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 musculusAdvantages-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 organismsSlime 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 regenerationLeech—study segmentation and neurobiologySea 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 squirtChick (Gallus gallus): Robust embryos, excellent for surgical manipulationOther fish: medaka, puffer fish (Fugu rubpripes, stripped down genome), goldfishOther mammals: rat (larger embryos than mouse), ferret (neurobiology)

Plus, “boutique” animals of particular evolutionary importance

Volvox Ascidian Chick

Genomes and Development. An Introduction to Developmental Biology.

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