Lecture 1 Overview of early mammalian development Tools for studying mammalian development...
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Lecture 1 Overview of early mammalian development Tools for studying mammalian development Fertilisation and parthenogenesis Mosaic vs regulated development
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
Slide 2
Embryogenesis in mammals occurs in utero - difficult to
observe. Important to study because of direct relevance for
understanding and treating disease. 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. Mouse is preferred model; Good genetics (inbred lines
etc), short generation time. Mammalian Development
Slide 3
Where am I?Who am I? Anterior (Head) Posterior (Tail) Ventral
(Back) Dorsal (Front) Left Right An anthropomorphic view of
development
Slide 4
In utero development in mouse occurs over 19-21 days
Slide 5
Preimplantation Development Trophectoderm Primitive (primary)
endoderm Inner cell mass Cleavage stages Zona pelucida Blastocoel
cavity Activation of embryonic genome Blastomere 01234 days
Slide 6
Early Post-implantation Development
Slide 7
Gastrulation and Beyond
Slide 8
Extraembryonic tissues
Slide 9
Experimental Tools for studying mouse embryos Embryological
approaches; Histological analysis and conventional microscopy Cell
fate mapping (dyes and now tagged loci) In vitro culture of
preimplantation stages and in some cases postimplantation
stages.
Slide 10
In situ hybridization Immunohistochemistry Eed + Nanog Oct4 +
Eed SectionsWholemount Embryological approaches; Gene expression
profiling of embryos, dissected fragments, derivative tissue
culture cell lines and single cells.
Slide 11
Chimera formation and embryo aggregation. Cell culture models
e.g. tetraploid chimeras for testing gene function in
extraembryonic vs embryonic lineages. Embryological approaches;
Embryonic stem (ES) cells
Slide 12
Genetic approaches; Classical mouse mutants Brachyury mouse
with short tail is dominant mutation in gene for transcription
factor required for mesoderm formation. Genetic screens Wild-type
and Nodal (d/d) mutant embryos with staining for markers of
primitive streak (brown) and ectoderm (dark blue). 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 SA SD Antibiotic
resistance marker Reporter gene IRES PolyA signal
Slide 13
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;
Slide 14
Transgene constructs; 100kb - Intact gene in BAC complete with
tissue specific regulatory sequences enhancerpromoter - Engineered
BAC with heterologous regulatory sequences, eg tetracycline
inducible - Plasmid with tissue specific regulatory sequences and
heterologous gene eg GFP reporter. Genetic approaches; Drawback;
high copy number gives non-physiological expression levels
Slide 15
Gene targeting in embryonic stem (ES) cells Genetic approaches;
X Homzygous mutants, double mutants etc Homzygous/double mutant ES
cells
Slide 16
Conventional gene knockout strategy (replacement vector)
Potential drawbacks are redundancy and lethality X X Positive
selectable Marker gene Negative selectable Marker gene Knock-out X
GFP Orf X Knock-in Genetic approaches;
Slide 17
Conditional gene knockout strategy; Bacterial site specific
recombinases (Cre-loxP or Flp-Frt) Genetic approaches;
Slide 18
Positive selectable Marker gene Negative selectable Marker gene
X X + site specific recombinase + Recombinase recognition sequence
Conditional gene knockout strategy; Genetic approaches;
Slide 19
Homozygous conditional allele Transgenic mouse expressing site
specific recombinase in tissue specific pattern 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. Conditional
gene knockout strategy; Genetic approaches;
Slide 20
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.
Slide 21
Pronuclear Maturation 12 24 Replication initiation M-phase hr
post fertilization 0 Second polar body Zona pelucida Maternal and
paternal genome remain separate (pronuclei) unitil first metaphase.
Male pronucleus. Female pronucleus. Syngamy
Slide 22
Parthenogenesis Limited viability suggests either that
sperm/fertilization confers essential properties for development or
that maternal genome alone is incapable of supporting development
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
Slide 23
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
Slide 24
Epigenesis vs Preformation
Slide 25
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. Mosaic and Regulated development
Slide 26
Tarkowski, (1959) Nature 184, p1286-7 2-cell embryo Donor
Recipient Regulated development in mouse embryos
Slide 27
Chimeras from aggregaton of 8-cell stage embryos 8-cell embryos
Remove zona pellucida Aggregate in dish Culture in vitro Chimeric
blastocyst Transfer to foster mother Chimeric progeny Tarkowski
(1961) Nature 190, 857-860
Slide 28
Chimeras from transfer of ICM cells Gardner later showed fate
of TE and PE is determined by blastocyst stage Gardner (1968),
Nature 220, p596-7