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

表观遗传修饰存在于细胞核中的组蛋白和DNA上，对胚胎发育至关重要。这些表观遗传标记有助于建立活性和抑制性染色质状态，这可以在细胞谱系中传播基因活性模式，还可以调节整体核结构和染色体功能。在小鼠胚胎发生中，核浓缩染色质的出现与分化和发育潜能的限制相一致，但其在这些过程中的因果关系尚不清楚。胚胎干细胞（embryonic stem cells，ES细胞）是由胚胎内细胞团（immune inner cell mass，ICM）分化而来的，具有染色质浓缩、高水平DNA甲基化和抑制性组蛋白修饰等特点，而ICM细胞则没有这些特点[1]。在胚胎干细胞中，这些通路是不稳定的，但已被提出参与从“血清状态”到“基态”的转换，被认为更接近地代表胚胎中的多能状态，以及在分化为其他细胞命运的过程中。我们假设表观遗传抑制通路可能在胚胎中的三维和信号背景下起作用，当ES细胞在培养皿中培养时，其可能较少受到支持或破坏。我们将使用各种实验和先进的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育，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志物和基因表达的影响进行比较。(ii)开发一种使用“胚胎样”重建方法的方案，将胚胎胎盘滋养外胚层干细胞与ES细胞混合，使细胞自组织成胚胎样结构[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作用非常相似的突变细胞是否在形成胚胎样组装体的三维和信号传导背景中显示出行为差异。(iii)使活成像报告子构建体适应表观遗传修饰以用于ES细胞和胚胎。(iv)建立新的成像方法，记录在一个培养皿中发生的多个胚胎样组装事件，基于光片显微镜的3D延时成像[3]。通过固定和免疫染色对干细胞和表观遗传标记进行进一步分析，这个博士项目涉及三个合作研究小组，将胚胎和干细胞表观遗传学的专业知识与先进光学物理和工程学的专业知识相结合，以揭示多能细胞中依赖于上下文的表观遗传信号机制和途径。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，这将改善干细胞在生物科学和生物医学中的应用。1. Wongtawan，T.，泰勒，J.E.，劳森，K.A.，威尔莫特岛，和Pennings，S.（2011年）。组蛋白H4 K20 me 3和HP 1alpha是发育中的晚期异染色质标记物，但存在于未分化的胚胎干细胞中。J Cell Sci 124，1878-1890.2.哈里森，S. E.，Sozen B.，Christodoulou N.，Kyprianou C.，泽尼卡-戈茨河（2017年）。胚胎干细胞和胚外干细胞体外组装模拟胚胎发生。科学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年5月;11（5）：541-4。doi：10.1038/nmeth.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.