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

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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.