The role of epigenetic pathways in defining pluripotent stem cell states in the embryo and ES cells

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Epigenetic modifications are found on the histones and DNA in the cell nucleus and are essential for embryo development. These epigenetic marks help to set up active and repressive chromatin states, which can propagate gene activity patterns in cell lineages but also regulate global nuclear architecture and chromosome function. The appearance of nuclear condensed chromatin in mouse embryogenesis coincides with differentiation and a restriction in developmental potential, but its causal involvement in these processes remains unclear. The derivation of embryonic stem (ES) cells from the embryo inner cell mass (ICM) results in their adoption of condensed chromatin, high levels of DNA methylation and repressive histone modifications that are not detectable in the ICM[1].Our project aims to gain understanding of the roles that epigenetic repression pathways play in the development of pluripotent cells in the mouse embryo and in cultured ES cells. In ES cells these pathways are dispensable but have been proposed to be involved in the conversion from 'serum state' to 'ground state', thought to more closely represent the pluripotent state in the embryo, as well as during differentiation to other cell fates.We hypothesise that epigenetic repression pathways may act in a three-dimensional and signalling context in the embryo, which may be less supported or disrupted when ES cells are cultured in a dish. We will explore this using a variety of experimental and advanced imaging approaches.The project aims to: (i) investigate the development of pluripotent ICM cells and other early cell fates in the preimplantation embryo, and the effects of inhibiting and downregulating epigenetic pathways on the formation of these cells. This will be compared with the effects of inhibition/down regulation of these pathways in cultured ES cells on cell markers and gene expression. (ii) develop a protocol using 'embryo-like' reconstitution methods that mix embryo placental trophectoderm stem cells with ES cells, allowing cells to self-organise into embryo-like structures[2]. Utilise this method for experiments mixing trophectoderm stem cells with existing mutant ES cell lines to record and determine whether mutant cells that act very similarly in a dish, display differences in behaviour in a 3-dimensional and signalling context in forming embryo-like assemblies.(iii) adapt live imaging reporter constructs for epigenetic modifications for use in ES cells and embryos.(iv) set up new imaging methodology to record multiple embryo-like assembly events occurring in a dish, based on 3D time lapse imaging by light sheet microscopy[3]. Embryo-like assemblies will be further analysed by fixation and immunostaining for stem cell and epigenetic markers.This PhD project involves three collaborating research groups, combining expertise in epigenetics in embryos and stem cells with expertise in advanced optical physics and engineering, to reveal context-dependent epigenetic signalling mechanisms and pathways acting in pluripotent cells. The research outcomes will provide more knowledge on the role of epigenetics in stem cells, which will lead to improved stem cell applications for bioscience and biomedicine. 1. Wongtawan, T., Taylor, J.E., Lawson, K.A., Wilmut, I., and Pennings, S. (2011). Histone H4K20me3 and HP1alpha are late heterochromatin markers in development, but present in undifferentiated embryonic stem cells. J Cell Sci 124, 1878-1890.2. Harrison, S.E., Sozen B., Christodoulou N., Kyprianou C., Zernicka-Goetz M. (2017). Assembly of embryonic and extraembryonic stem cells to mimic embryogenesis in vitro. Science. 356, (6334) doi: 10.1126/science.aal18103. Vettenburg T, Dalgarno HI, Nylk J, Coll-Llado C, Ferrier DE, Cizmar T, Gunn-Moore FJ, Dholakia K.Nat Methods. 2014 May;11(5):541-4. doi: 10.1038/nmeth.2922.