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Lecture 1
• Overview of early mammalian development
• Tools for studying mammalian development
• Fertilisation and parthenogenesis
• Mosaic vs regulated development
You should understand
• Non-equivalence of maternal and paternal genomes
• Mammalian development is highly regulated
Mammalian Development
• Embryogenesis in mammals occurs in utero - difficult to observe.
• Important to study because of direct relevance for understanding and treating disease.
• Mouse is preferred model;
Good genetics (inbred lines etc), short generation time.
Isolation of tissue culture models, e.g embryonic stem cells, is relatively easy.
Also highly advantageous for genetic manipulation, knock-out, knock-in etc
- Functional genomics studies
- Disease models for basic science and pharmacology.
An anthropomorphic view of development
Who am I?
Where am I?
Anterior (Head)
Right
Ventral (Back)
Dorsal (Front)
Left
Posterior (Tail)
In utero development in mouse occurs over 19-21 days
Preimplantation Development
0
1
2
Cleavage stages
3
4
days
Primitive (primary) endoderm
Blastocoel cavity
Zona pelucida
Blastomere
Activation of embryonic genome
Trophectoderm
Inner cell mass
Early Post-implantation Development
Gastrulation and Beyond
Extraembryonic tissues
Experimental Tools for studying mouse embryos
Embryological approaches;
• Histological analysis and conventional microscopy
• In vitro culture of preimplantation stages and in some cases postimplantation stages.
• Cell fate mapping (dyes and now tagged loci)
Embryological approaches;
• Gene expression profiling of embryos, dissected fragments, derivative
tissue culture cell lines and single cells.
•
In situ hybridization
Sections
•
Wholemount
Immunohistochemistry
Oct4 + Eed
Eed + Nanog
Embryological approaches;
• Chimera formation and embryo aggregation.
e.g. tetraploid chimeras for
testing gene function in
extraembryonic vs
embryonic lineages.
• Cell culture models
Embryonic stem (ES) cells
Genetic approaches;
• Classical mouse mutants
Brachyury mouse with short tail is dominant mutation in gene for
transcription factor required for mesoderm formation.
• Genetic screens
Chemical (ENU) mutagenesis – requires lengthy genetic mapping and cloning to identify
mutated locus
Insertional or ‘gene trap’ mutagenesis in ES cells – can go directly to gene of interest
Antibiotic resistance
marker
IRES
Reporter gene
PolyA signal
SA
SD
Wild-type and Nodal (d/d) mutant embryos with
staining for markers of primitive streak (brown)
and ectoderm (dark blue).
Genetic approaches;
• Production of transgenic mice
- Gene construct injected into male pronucleus of 1-cell embryo
- DNA integrates randomly into the genome
- Usually at single site but in multiple copies
- Resulting mice can be bred and then maintained
by monitoring continued presence of the transgene using PCR etc.
- Gene construct can be assembled in plasmid (up to 25kb) or bacterial artificial chromosome (BAC)
vectors (100-200kb).
Genetic approaches;
Transgene constructs;
- Intact gene in BAC complete with tissue specific regulatory sequences
100kb
enhancer
promoter
- Engineered BAC with heterologous regulatory sequences, eg tetracycline inducible
- Plasmid with tissue specific regulatory sequences and heterologous gene eg GFP reporter.
Drawback; high copy number gives non-physiological expression levels
Genetic approaches;
• Gene targeting in embryonic stem (ES) cells
Homzygous/double
mutant ES cells
X
Homzygous mutants,
double mutants etc
Genetic approaches;
Conventional gene knockout strategy (replacement vector)
Positive selectable
Marker gene
Negative selectable
Marker gene
X
X
Knock-in
GFP
Orf
X
Knock-out
X
Potential drawbacks are redundancy and lethality
Genetic approaches;
Conditional gene knockout strategy;
Bacterial site specific recombinases (Cre-loxP or Flp-Frt)
Genetic approaches;
Conditional gene knockout strategy;
Recombinase recognition sequence
Positive selectable
Marker gene
X
Negative selectable
Marker gene
X
+ site specific recombinase
+
Genetic approaches;
Conditional gene knockout strategy;
Transgenic mouse expressing
site specific recombinase
in tissue specific pattern
Homozygous conditional allele
X
Analyse phenotype in F1 embryos or adults
Examples of recombinase driver transgenics;
- Cre recombinase driven by Nanog promoter
- Estrogen receptor-Cre recombinase fusion driven by constitutive promoter.
Addition of Tamoxifen to drinking water triggers nuclear
translocation of recombinase giving temporal control of gene deletion.
Fertilisation
• Penetration of cumulus cells
• Acrosomal reaction penetrates zona pellucida made up of glycoproteins
• Sperm and egg plasma membranes fuse and sperm nucleus enters egg.
• Fertilization triggers dramatic release of calcium in the egg, setting in train completion of
female meiosis etc.
Pronuclear Maturation
Second polar body
Zona pelucida
Female pronucleus.
Syngamy
Male pronucleus.
12
0
24
hr post fertilization
Replication
initiation
•
M-phase
Maternal and paternal genome remain separate (pronuclei) unitil first metaphase.
Parthenogenesis
Parthenogenetic activation
- Genetic background
- In vitro manipulation
- Pronase/hyalouronidase
- Heat shock
- Ethanol
- Strontium chloride
• Oocytes can be activated in the absence of fertilization, leading to parthenogenetic development
• Parthenogenetic embryos have limited viability, contrasting with other model organisms
• Limited viability suggests either that sperm/fertilization confers essential properties for development or
that maternal genome alone is incapable of supporting development
Non-equivalent contribution of maternal and paternal genomes
Recipient zygote
?
Donor zygote
Barton, Surani , Norris (1984)
Nature 311, p374-6
McGrath and Solter, (1984)
Cell 37, p179-183
• Gynogenetic embryos have retarded growth/development of extraembryonic tissues
• Androgenetic embryos have retarded growth/development of embryonic tissues
Epigenesis vs Preformation
Mosaic and Regulated development
•
Roux (1888) shows ‘mosaic development’ of frog embryo following ablation of one cell in
two-cell embryo – formation of ‘half’ embryo.
•
Driesch (1895) finds opposite is true for sea urchin, normal albeit smaller embryo develops
from one of two cells – ‘regulated development’.
Regulated development in mouse embryos
Donor
Recipient
2-cell
embryo
Tarkowski, (1959)
Nature 184, p1286-7
Chimeras from aggregaton of 8-cell stage embryos
8-cell embryos
Remove zona pellucida
Aggregate in dish
Culture in vitro
Transfer to foster mother
Chimeric blastocyst
Chimeric progeny
Tarkowski (1961) Nature 190, 857-860
Chimeras from transfer of ICM cells
Gardner (1968), Nature 220, p596-7
• Gardner later showed fate of TE and PE is determined by blastocyst stage
End lecture 1