1. 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

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

3. Where am I?Who am I? Anterior (Head) Posterior (Tail) Ventral (Back) Dorsal (Front) Left Right An anthropomorphic view of development

4. In utero development in mouse occurs over 19-21 days

5. Preimplantation Development Trophectoderm Primitive (primary) endoderm Inner cell mass Cleavage stages Zona pelucida Blastocoel cavity Activation of embryonic genome Blastomere 01234 days

6. Early Post-implantation Development

7. Gastrulation and Beyond

8. Extraembryonic tissues

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.

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.

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

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

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;

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

15. Gene targeting in embryonic stem (ES) cells Genetic approaches; X Homzygous mutants, double mutants etc Homzygous/double mutant ES cells

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;

17. Conditional gene knockout strategy; Bacterial site specific recombinases (Cre-loxP or Flp-Frt) Genetic approaches;

18. Positive selectable Marker gene Negative selectable Marker gene X X + site specific recombinase + Recombinase recognition sequence Conditional gene knockout strategy; Genetic approaches;

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;

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.

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

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

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

24. Epigenesis vs Preformation

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

26. Tarkowski, (1959) Nature 184, p1286-7 2-cell embryo Donor Recipient Regulated development in mouse embryos

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

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

29. End lecture 1