Lecture 1 Overview of early mammalian development Tools for studying mammalian development

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. 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.

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

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

5. 0 1 2 3 4 Preimplantation Development days Cleavage stages Primitive (primary) endoderm Blastocoel cavity Blastomere Inner cell mass Zona pelucida Trophectoderm Activation of embryonic genome

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 • In vitro culture of preimplantation stages and in some cases postimplantation stages. • Cell fate mapping (dyes and now tagged loci)

10. Embryological approaches; • Gene expression profiling of embryos, dissected fragments, derivative tissue culture cell lines and single cells. • In situ hybridization Sections Wholemount • Immunohistochemistry Eed +Nanog Oct4 +Eed

11. 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

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 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 Reporter gene PolyA signal IRES SA Wild-type and Nodal (d/d) mutant embryos with staining for markers of primitive streak (brown) and ectoderm (dark blue). SD

13. 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).

14. 100kb enhancer promoter Genetic approaches; Transgene constructs; - Intact gene in BAC complete with tissue specific regulatory sequences - 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

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

16. Knock-out Negative selectable Marker gene Positive selectable Marker gene X X Knock-in GFP Orf X X Genetic approaches; Conventional gene knockout strategy (replacement vector) Potential drawbacks are redundancy and lethality

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

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

19. 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.

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 Second polar body Zona pelucida Syngamy Male pronucleus. Female pronucleus. 12 24 0 hr post fertilization Replication initiation M-phase • Maternal and paternal genome remain separate (pronuclei) unitil first metaphase.

22. 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

23. Recipient zygote Donor zygote Non-equivalent contribution of maternal and paternal genomes ? 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. 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’.

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

27. 8-cell embryos Remove zona pellucida Aggregate in dish Culture in vitro Transfer to foster mother Chimeric blastocyst Chimeric progeny Chimeras from aggregaton of 8-cell stage embryos Tarkowski (1961) Nature 190, 857-860

28. 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

29. End lecture 1