Visualizing nanoscale 3D genome architecture and transcriptional state during cell fate specification in the early mouse embryo

哺乳动物的发育是一个高度可塑的过程，从精子使卵母细胞受精形成受精卵开始，受精卵是一个二倍体的全能性细胞，含有两个前核，经过几次快速的细胞分裂，形成一个囊胚，能够植入母亲的子宫壁。囊胚包含哺乳动物发育过程中最初的两种谱系细胞类型，胚胎外滋养外胚层和提供胚胎干细胞的内细胞群。它们具有不同的形态和不同的表达谱。该领域最近开始了解到，除了转录组和表观基因组的许多变化外，早期分化步骤的调节还与染色质分层空间组织的主要变化有关。然而，基因组位点的空间重组是否或如何影响其活性，反之亦然，以及空间基因组结构的变化在谱系之间的差异以及在谱系规范过程中如何调节基因表达仍有待研究。我们知识上的这一差距很大程度上是由于缺乏对单个分化细胞中基因组的物理3D结构和转录活性的直接组合可视化，这将使我们能够揭示空间基因组结构的变化何时以及如何与单个胚胎细胞内原位基因表达等功能的变化相关联。在我们提出的项目中，我们计划解决我们知识上的这一空白，并破译早期小鼠胚胎单细胞基因组结构和转录之间的关系。为了实现这一目标，我们将把我们最近开发的3D染色质追踪技术与单等位基因转录活性和核结构的成像结合起来，并将这些与细胞命运联系起来。这种新颖的方法将使我们能够定量地绘制当相同的姐妹细胞分化为内细胞群和滋养外胚层时基因组结构的变化。因此，我们的实验将揭示基因组的哪些结构特征是哺乳动物生命中第一个命运规范的基础。总之，该项目将首次在纳米尺度上直接观察哺乳动物早期发育过程中单个卵裂球中与转录和细胞命运相关的基因组结构变化。结合联盟中可用的单细胞转录组学和活细胞成像技术，这将使我们能够创建基因组，转录组和发育中的胚胎命运规范之间结构-功能关系的完整视图。

Mammalian development is a highly plastic process that begins with fertilisation of the oocyte bythe sperm to form the zygote, a diploid totipotent cell containing two pro-nuclei, which undergoes several rapid cell divisions to build a blastocyst that is competent for implantation into the uterine wall of the mother. The blastocyst contains the first two lineage-committed cell types in mammalian development, the extraembryonic trophectoderm and the inner cell mass that provides embryonic stem cells. These have different morphologies and differential expression profiles. The field has recently started to understand that regulation of early differentiation steps is associated with major changes in the hierarchical spatial organization of chromatin, in addition to many changes in the transcriptome and the epigenome. However, it remains to be investigated whether or how the spatial restructuring of a genomic locus affects its activity, or vice versa, and how the changes in spatial genome architecture differ between lineages and modulate gene expression during lineage specification. This gap in our knowledge is largely due to the fact that direct combined visualization of the physical 3D structure of the genome and transcriptional activity in single differentiating cells is lacking, which would allow us to reveal when and how changes in the spatial genome architecture are linked to changes in function such as gene expression, in situ inside single embryonic cells. In the proposed project we plan to address this gap in our knowledge and decipher the relation between genomic architecture and transcription in single cells of the early mouse embryo. To achieve this, we will combine our recently developed 3D chromatin tracing technology with imaging of single-allele transcriptional activity and nuclear architecture and relate these to cellular fate. This novel approach will allow us to quantitatively map how genome architecture changes when identical sister cells differentiate into inner cell mass and trophectoderm. Our experiments will thus reveal which structural hallmarks of the genome underlie the first fate specification in mammalian life. In summary, the proposed project will for the first time directly visualise changes in genome architecture associated with transcription and cell fate at the nanoscale in single blastomeres during early mammalian development.In combination with the single-cell transcriptomics and live-cell imaging technologies available within the consortium, this will allow us to create a complete view of the structure-function relationship between genome, transcriptome and fate specification in the developing embryo.