Soon after implantation, the mammalian embryo needs to instruct the specification of the epiblast precursors required for the formation of the foetal tissues. At the exit from pluripotency, global epigenetic and transcriptional remodelling occurs. These changes are essential for gastrulation, the process by which all three germ layers - ectoderm, mesoderm, and endoderm - are specified. Recent advances in single-cell sequencing technologies have allowed the characterisation of the transcriptional and epigenetic changes during mouse gastrulation and early organogenesis. However, the precise molecular mechanisms that control cell fate decisions are still poorly understood. Therefore, my PhD project aimed to understand further how genetically identical cells can be primed for differentiation and specification. My PhD work's main focus was to characterise (1) the role of the spatial environment and (2) DNA methylation in cell lineage priming and specification during embryonic development.

To better understand the spatial environment's role on cell fate specification (Aim 1), we used the image-based single-cell transcriptomics method, seqFISH, to precisely measure the mRNA abundance of 387 carefully selected target genes in mouse embryos. Therefore, I developed a new cell segmentation strategy and performed seqFISH on E8.5 mouse embryo tissue sections to characterise the roles of the spatial environment on cell fate specification during organogenesis. Joint analysis of the seqFISH data with previously published scRNA-seq data allowed us to address biological questions related to spatial coherence of cell types, the emergence of the midbrain-hindbrain boundary and gut tube organogenesis. Strikingly, we demonstrated that the spatial patterning of the gut tube is associated with distinct organ primordia. The spatial atlas uncovers axes of resolution that cannot be reconstructed from single-cell RNA sequencing data, for example, we observe the dorsal-ventral separation of oesophageal and tracheal progenitor populations 24-hours earlier than previously appreciated.

The manipulation of DNA methylation levels by knockout of methylation modifiers in vivo results in embryonic lethality shortly after gastrulation, highlighting the essential role of fine-tuned DNA methylation in mammalian development. However, how DNA methylation influences cell fate decisions is still poorly understood. To address this, I used deletion of DNA methylation enzymes in mouse embryos and stem cell culture systems to ask whether DNA methylation is required for cell fate choices (Aim 2). I showed that DNA methylation is necessary to exit pluripotency and restrict the differentiation potential to extraembryonic lineages. Notably, we showed that the loss of TET dioxygenases results in mesodermal differentiation defects, which was particularly pronounced for mature blood cells and cardiomyocytes. Strikingly, we demonstrated that TETs are required to demethylate and activate mesoderm specific enhancers during cellular differentiation, using single-cell nucleosome, methylome and transcriptome sequencing (scNMT-seq). Hence, we hypothesised that the inability of the cells to reprogramme the epigenome causes the differentiation defect detected. The results demonstrate the essential role of active DNA demethylation for lineage specification. We are currently expanding on these intriguing observations by identifying lineage-specific DNA methylation defects in the blood lineage of Tet1/2/3 TKO chimeras. The results will reveal core functions of DNA demethylation during cell lineage specification and establishment of developmental competence.

In sum, my PhD project provides a detailed study of the role of the spatial environment on transcriptional regulation (Aim 1) and the causal relationship between DNA methylation and cell fate in early mammalian embryogenesis (Aim 2).