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

表观遗传修饰是在细胞核中的组蛋白和DNA上发现的，对胚胎发育至关重要。这些表观遗传标记有助于建立活跃和抑制染色质状态，染色质状态可以在细胞谱系中传播基因活性模式，但也调节全球核结构和染色体功能。小鼠胚胎发育中核染色质的出现与分化和发育潜力的限制是一致的，但它与这些过程的因果关系尚不清楚。从胚胎内细胞团(ICM)衍生的胚胎干细胞(ES)导致它们采用浓缩的染色质、高水平的DNA甲基化和抑制性组蛋白修饰，这些在ICM中是检测不到的[1]。我们的项目旨在了解表观遗传抑制途径在小鼠胚胎和培养的ES细胞中多能细胞的发育中所起的作用。在ES细胞中，这些通路是可有可无的，但已被认为参与了从血清状态到基态的转换，被认为更能代表胚胎的多能性状态，以及向其他细胞脂肪的分化。我们假设，表观遗传抑制通路可能在胚胎的三维和信号环境中发挥作用，当ES细胞在培养皿中培养时，这种作用可能得到较少的支持或破坏。我们将使用各种实验和高级成像方法来探索这一点。该项目旨在：(I)研究植入前胚胎中多能ICM细胞和其他早期细胞命运的发展，以及抑制和下调表观遗传途径对这些细胞形成的影响。这将与在培养的ES细胞中抑制/下调这些途径对细胞标记物和基因表达的影响进行比较。(Ii)开发一种方案，使用“类胚胎”重建方法，将胚胎胎盘滋养外胚层干细胞与胚胎干细胞混合，使细胞能够自组织形成类胚胎结构[2]。利用这种方法进行实验，将滋养外胚层干细胞与现有的突变ES细胞系混合，以记录和确定在培养皿中行为非常相似的突变细胞是否在形成类似胚胎的组装的三维和信号环境中表现出不同的行为。(Iii)使活体成像报告构建物用于用于ES细胞和胚胎的表观遗传修饰。(Iv)建立新的成像方法，基于光片显微镜的3D时间推移成像，记录在培养皿中发生的多个类似胚胎的组装事件。胚胎样组件将通过干细胞和表观遗传标记的固定和免疫染色进一步分析。这个博士项目涉及三个合作的研究小组，将胚胎和干细胞的表观遗传学专业知识与高级光学物理和工程专业知识相结合，以揭示与环境相关的表观遗传信号机制和作用于多能细胞的途径。研究成果将提供更多关于表观遗传学在干细胞中的作用的知识，这将导致干细胞在生物科学和生物医学中的应用得到改善。1.Wongtawan，T.，Taylor，J.E.，Lawson，K.A.，Wilmut，I.和Pennings，S.(2011年)。组蛋白H4K20me3和HP1pha是发育中的晚期异染色质标记，但存在于未分化的胚胎干细胞中。《细胞科学》第124期，1878-1890.2。哈里森，S.E.，Sozen B.，Christodoulon.，Kyprianou C.，Zericka-Goetz M.(2017)。在体外组装胚胎和胚胎外干细胞以模拟胚胎发生。科学。Doi：10.1126/cience.aal18103.[2]Vettenburg T，Dalgarno HI，Nylk J，Coll-Llado C，Ferrier DE，Cizmar T，Gunn-Moore FJ，Dholaki K.NAT方法。2014年5月；11(5)：541-4。DOI：10.1038/nmet.2922.

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.