1. Pre-implantation Development Dr Rachel Gibbons

2. Outline How to study mammalian embryos? How are they different from non-mammalian embryos? Why are these early stages so important, what happens? Morphological, protein, mRNA and DNA changes APPLICATIONS IVF, PGD, stem cell derivation and differentiation, transgenic animals

3. From ‘Manipulating the Mouse embryo’, Hogan, Beddington, Constantini and Lacy

5. Technologies needed to study pre- implantation development Reliable embryo culture techniques (1960’s Biggers et al) Embryo transfer to allow replacement of cultured pre-implantation embryos in pseudo pregnant females, to evaluate media by number of live pups born.

6. Differences between mammalian and non mammalian development Xenopus and drosophila have a pre –patterned development and if this is disturbed development will fail At the earliest stages of mammalian development all cleavage stage blastomeres (cells) are equivalent and can be removed without developmental failure.

7. Early human development in vitro Late Day 1 2- cells Day 1 Day 2 4-cellsDay 3 8-cells Day 4 morula Blastocyst Day 5 Hatching Day 6 Hatched Late Day 6 ICM Day 4 ICM

9. Developmental control is initially directed by maternal proteins and mRNAs stored in the egg. Some mRNAs critical for development will have been stored as “masked” mRNAs in the egg and will only be translated after fertilisation has occurred. The maternal to zygotic transition (MZT) then occurs, switching control from the maternal to the embryonic genome. In mouse this occurs by the 2 cell stage. In humans this occurs at the 4-8 cell stage

10. MZT is essential to the continued development of the embryo. Firstly, maternal mRNAs must be degraded. Secondly, transcripts that are common to the oocyte and embryo e.g. actin must be replaced by the embryonic genome Thirdly, the dramatic reprogramming of gene expression must occur that will result in novel mRNAs being expressed in the embryo that were not expressed in the oocyte. The genes involved and molecular mechanisms of this genetic reprogramming are the subject of much research e.g “knockout mice”

11. Parental imprinting “Parental (genomic) imprinting is the process by which the differential expression of maternal and paternal alleles at certain loci in mammalian embryos occurs.” (Moore & Reik 1996). Relatively few imprinted genes exist in the genome (perhaps 100 out of a total of 30,000 genes) Organised into clusters known as imprinting centres (ICRs). The expression of these genes is controlled by the DNA methylation status of particular cytosine-guanine dinucleotides within them. DNA methylation patterns are regulated in part by DNA methyltransferase enzymes eg Dnmt 1 maintains the existing methylation pattern during DNA replication. The correct expression of imprinted genes is vital for embryo/foetal development, as illustrated by the following experiment…

12. Classic pronuclear transfer experiments (e.g.Barton et al. 1984) MaleFemale Enucleated recipient eggs Reconstructed embryos Androgenetic. Embryos retarded, trophoblast well developed. No development to term. Gynogenetic. Meagre extraembryonic tissue. No development to term. Normal. 40-50% of constructs developed to term Pronuclei 1 male and 1 female

13. Imprinting Diseases Beckwith-Wiedemann Syndrome (BWS), Prader-Willi Syndrome (PWS) and Angelman Syndrome (AS) are all caused by errors in genomic imprinting. PWS and AS are both caused by absence of the same region of chromosome 15 (15q11.2-q13). This is either due to a de novo deletion or uniparental disomy. The symptoms of the two syndromes are quite different and this is due to which copy is lacking –PWS is caused by a lack of the paternal copy. –AS is caused by a lack of the maternal copy. BWS is caused by the lack of a maternal copy of a region of chromosome 11 (11p15.5) that contains at least 15 genes many of which many are imprinted

14. Special characteristics of mammalian embryos No pre-patterning in egg and early embryo Inner cell mass cells are pluripotent Pre-implantation embryos can be manipulated without compromising subsequent development.

15. Diagnostic and Therapeutic Technologies

16. Assisted Reproduction Techniques (ART)

17. IVF ICSI Human preimplantation Development day 0-2 Fertilised egg 2 cell embryo 4 cell embryo

18. Pre-implantantion Genetic Diagnosis (PGD)

19. Technique of PGD

20. Embryo Biopsy

22. Regenerative Stem Cell Therapy

24. Current Clinical Trials Using ES cells Two clinical trails using human derived ES cells are currently being carried out at UCLA, USA to test whether these cells can be used to treat two incurable eye disorders. –Dry age-related macular dystrophy (AMD) is the commonest form of blindness in the developed world and leads to vision loss in people aged 55 or over. –Stargardt’s macular dystrophy is an inherited condition and usually develops between the ages of 10 and 20 Both conditions are caused by the progressive loss of retinal pigment epithelial (RPE) cells, which support, protect and nourish the light sensitive cells that provide vision. The technology, developed by Advanced Cell Technology in Massachusetts, has been reported to have promising results in mice and rats.

25. Current Clinical Trials Using ES cells Patients in the trials will receive injections of ES cells programmed to behave like RPE cells into the eye at varying doses. The first aim is to test the safety and tolerability of the injections and to see whether progression of the disease can be slowed. If successful, the aim would then be to carry out future trials on larger numbers of patients with the aim of halting or reversing the disease. The study will have a two year follow-up and should be complete in July 2014 A clinical trail using ES cells aiming to treat spinal cord injury was started in 2010. This again aimed to test the safety of the treatment and was used on 4 patients, with no reported negative outcomes. However, due to economic reasons, the company running the trials (Geron) has discontinued it.

26. Human-animal hybrid embryos Donor cell e.g. fibroblast Egg cell Nucleus containing genetic Information is removed Nucleus containing genetic Information is removed Egg with inserted genetic information from donor cell. Mitochondria from the egg cell remains Following activation cell division begins and an embryo develops. The majority of the genetic information is from the donor cell (nuclear DNA and a small amount of genetic information is from the egg cell (mitochondrial DNA). Mitochondria Genetic information If a blastocyst forms, the inner cell mass can be removed and used to create an embryonic stem cell line Inner Cell Mass Trophectoderm Blastocyst Embryonic stem cells can be used for research.

28. In conclusion... The study of mammalian embryo development and IVF relied on first developing effective culture techniques. Techniques that have since been developed, such as PGD, embryonic stem cell growth, transgenic animals etc rely on being able to manipulate the mammalian embryo The ability to manipulate mammalian embryos in vitro relies on –the lack of pre-patterning in the egg and early embryo –the pluripotency of the cells within the early embryo.

29. References Allegrucci C., Thurston, A., Lucas, E. and Young, L (2005) Epigenetics and the germline. Reproduction 129:137-149 Barton, S.C., Surani, M.A. and Norris, M.L. (1984) Role of paternal and maternal genomes in mouse development. Nature 311: 374-376 Nichols, J. (2001) Introducing embryonic stem cells. Current Biology 11R503-505 Wang, H. and Dey, S.K. (2006) Roadmap to embryo implantation: clues from mouse models. Nature Reviews Genetics 7: 185-199 Schultz, R.M. (2002) The molecular foundations of the maternal to zygotic transition in the pre-implantation embryo. Human Reproduction Update 8: 323-331