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