Download - Lecture 1 Overview of early mammalian development Tools for studying mammalian 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
• 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
Where am I?Who am I?
Anterior (Head)
Posterior (Tail)
Ventral (Back)Dorsal (Front)
Left
Right
An anthropomorphic view of development
In utero development in mouse occurs over 19-21 days
Preimplantation Development
Trophectoderm
Primitive (primary) endoderm
Inner cell mass
Cleavage stages
Zona pelucida
Blastocoel cavity
Activation of embryonic genome
Blastomere
0 1 2 3 4 days
Early Post-implantation Development
Gastrulation and Beyond
Extraembryonic tissues
Experimental Tools for studying mouse embryosEmbryological 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.
• In situ hybridization
• Immunohistochemistry
Eed + NanogOct4 + Eed
Sections Wholemount
Embryological approaches; • Gene expression profiling of embryos, dissected fragments, derivative tissue culture cell lines and single cells.
• 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
Genetic approaches;
• Classical mouse mutants
Brachyury mouse with short tail is dominant mutation in gene fortranscription 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 resistancemarker
Reporter gene
IRES PolyA signal
• 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 maintainedby 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;
100kb
- Intact gene in BAC complete with tissue specific regulatory sequences
enhancer promoter
- 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
• Gene targeting in embryonic stem (ES) cells
Genetic approaches;
XHomzygous mutants,double mutants etc
Homzygous/double mutant ES cells
Conventional gene knockout strategy (replacement vector)
Potential drawbacks are redundancy and lethality
X X
Positive selectableMarker gene
Negative selectableMarker gene
Knock-out
X
GFP Orf
X
Knock-in
Genetic approaches;
Conditional gene knockout strategy;
Bacterial site specific recombinases (Cre-loxP or Flp-Frt)
Genetic approaches;
Positive selectableMarker gene
Negative selectableMarker gene
X X
+ site specific recombinase
+
Recombinase recognition sequence
Conditional gene knockout strategy;
Genetic approaches;
Homozygous conditional allele Transgenic mouse expressing site specific recombinasein 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 nucleartranslocation of recombinase giving temporal control of gene deletion.
Conditional gene knockout strategy;
Genetic approaches;
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
12 24
Replicationinitiation
M-phase
hr post fertilization0
Second polar body
Zona pelucida
• Maternal and paternal genome remain separate (pronuclei) unitil first metaphase.
Male pronucleus. Female pronucleus.
Syngamy
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
Non-equivalent contribution of maternal and paternal genomes
?Recipient zygote
Donor zygote
Barton, Surani , Norris (1984)Nature 311, p374-6McGrath 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
• 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
Tarkowski, (1959)Nature 184, p1286-7
2-cellembryo
Donor
Recipient
Regulated development in mouse embryos
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
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
End lecture 1