Download An Introduction to Model Organisms of Development

An Introduction
to Model
Organisms of
Development
What makes a good model organism?
Gas: 2000 liters of methane gas released/day!
What makes a good model organism?
Size : 6 tons
250kg food eaten every
100kg of elephant dung/day
Gestation : 23 months
Females give birth to single
offspring every five years
Sexual maturity at age 12
Gas: 2000 liters of methane gas released/day!
Size : 1 mm in length
Live on a diet of bacteria
Gestation : 500,000
offspring in 1 week from
single organism
Sexual maturity in 3 days
Genome : Sequenced!
Homo sapiens
Mammals
Mus musculus
Amphibians
Arthropods
Xenopus laevis
Drosophila
melanogaster
Nematodes
Caenorhabditis
elegans
Caenorhabditis elegans
Nematode Worm
Nematodes account for an estimated four of
every five animals in the world !
Smooth-skinned, unsegmented worms first used as a model organism by Sydney
Brenner in 1965
C. elegans is diploid and has five pairs of autosomal chromosomes (named I, II,
III, IV and V) and a pair of sex chromosomes (X).
Most adults are hermaphrodite (XX) but .05% of lab populations are male (XO)
Lifespan is 2 to 3 weeks
Worms are usually kept on petri plates and fed E.coli
About 10,000 worms fit on a single plate
Developmental fate of every cell is known
in C. elegans
8 to 17 rounds of division are required for cell differentiation
depending on tissue type and cell function
959 Somatic cells
- all visible with microscope
- 300 neurons
- 81 muscle cells
- 131 cells undergo
programmed cell death
• C. elegans genome contains 19,000 genes and is fully sequenced
• 70% of human genes have worm homologues
• C. elegans will be the first and possibly only animal that we know
everything about at a cellular and molecular level
• Gene function studies have become relatively simple with the
recent discovery of RNAi
Using RNA interference for local and systemic
gene silencing in C. elegans
A.
A. C. elegans hermaphrodite expressing
GFP transgenes in the pharynx and the
nuclei of body-wall muscle cells
B.
B. C. elegans hermaphrodite expressing
GFP transgenes + GFP double stranded
RNA in the pharynx
Small RNAs as regulate gene expression during development
• Look for heterochronic defects in mutagenesis screen
-- cells behave as if in an earlier or later developmental stage
• Regulatory cascades unveiled which involve small RNAs (21-22 nts)
• Lin-4 and Let-7 encode short untranslated RNAs and function by
binding to complementary sequences in mRNAs of specific genes
controlling development
• Lin-4 expression allows cells to progress from larval stage 1 to 3
• Let-7 expression allows cells to progress from late larval to adult stages
Developmental mRNA
Lin-4
No translation of mRNA
Molecular genetic of life-span in C. elegans
C. elegans can live 2X longer under some experimental
conditions: Mutations in DAF-16, AGE-1, DAF-2, or CLK-1;
heat shock proteins; feeding behavior; free radical exposure
and oxidative stress
Pathways involved appear to be connected
General features of longer life span include less reproduction,
less growth and more DNA/cellular repair
Could be related to caloric restriction observations in mammals
Drosophila melanogaster
Common Fruit Fly
Most studied animal model
Life Cycle of 2 weeks (fertilization to sexual maturity)
Four pairs of chromosomes: the X/Y sex chromosomes and the
autosomes 2,3, and 4
14,000 Genes, sequenced genome and 2/3 of human disease
genes have fly homologues
Large repositories of mutant flies available
Conservation of patterning between
flies and mammals
In situ hybridization of whole embryo can reveal
patterns of gene expression during development
RNA or DNA probes and labeled antibodies are used.
Polytene Chromosomes
• Present in salivary glands of flies
• Originate from chromosomal
duplication with no cell division
• Have patterns of dark and light
bands unique for each
chromosomal section visible with a
light microscope
• Can be labeled with nucleic acid
probes
• Can be used to determine binding
site of labeled proteins
• Chromosomal rearrangments and
deletions can be visualized
Wildtype fly
Antennapedia mutant: Antenna are
transformed into metathoracic (second
second thoracic segment) legs
Studying Organogenesis in Drosophila
Imaginal discs are groups of undifferentiated cells in larva that give rise
to adult organs and structures
Transplantation and gene mis-expression studies allow characterization
of organ formation at cell and molecular level
Organ-specific genes have mammalian homologues
Xenopus Laevis
African clawed toad
Advantages of using Xenopus as a model:
• Vertebrate model with fundamental features of land-dwelling
vertebrates
• Oocytes are large and undergo external development
• Females can be stimulated to ovulate with hormones
•Development is rapid; fertilization to fully formed tadpole in a
few days
.
Large size allows study of movement of cells
within Xenopus embryos
• Cleavage every 30 minutes
• Gastrulation at 10 hours
• 1 day to neurulation
• Germ layers and structural
characteristics are easily
observed
• Manipulation of embryo may
involve surgery or mRNA
injection
Study of Xenopus Development by Embryo Injections
• mRNAs and cDNAs can be injected to study
role of genes and proteins
• Antisense to knockout expression
• Over- and mis-expression of protein of
interest
• Alternate protein forms: dominant negative,
constitutively active, etc.
• Reporter forms (GFP, etc.)
• Study maternal vs zygotic contributions
• Signaling molecules and chemical agents can
be applied to determine affects on development
The Embryonic Signaling Center:
Spemann’s Organizer
• Classic experiment first performed by Spemann
and Mangold in 1924
• Grafted dorsal lip of an embryo onto a second
embryo
• Gastrulation initiated at both sites
• Second whole set of body structures formed
Cell fate studies in Xenopus: Noggin
• Noggin expression permits
cells to become brain and
nervous system tissue
• No Noggin expression
results in tissue becoming
skin, bone
• Noggin is an inhibitor of
BMPs which promote bone
growth
• Use nucleic acid
microinjection to knockout or
over-express noggin
Mus Musculus
House Mouse
• Best model for mammalian development
• Life cycle approximately 9 weeks; 21 day gestation
• Litters up to 20 pups
• Genome sequenced
• Many inbred strains characterized (450 available)
• Genetic manipulations well developed
Embryos can be perturbed in various ways but
give rise to normal mouse
• Allows genetic manipulation of embryo possible
• Early embryo can be split to yield two “twins”
• Two morulas can be combined to form a chimera
• Cells from an embryo can be injected into another blastocyst to form a
chimera
Mouse embryos as a source of embryonic
stem cells
• Culture inner cell mass (gives rise
to whole embryo)
• ES cells will divide indefinitely
without differentiating is cultured
appropriately
• ES cells are totipotent; adult stem
cells tend to be pluripotent
•Studying ES cells could lead to human therapies for various
diseases
• ES cells good for genetic manipulation since whole mouse can be
obtained after injection into blastocyst
Mouse embryo with Hox gene marker
(created using methods described)
Motivations for understanding development: The
cancer connection
• Many human disease gene
homologues are required for
development
• Cancer results in dedifferentiation
of cells: development in “reverse”
• Embryonic lethality of knock-out
mice has led to concentration on
understanding mammalian
development
• There are ways around lethality
for studying gene function
Egfr knockout: contribution of
genetic background