Investigating the role of Gata3 in the endothelial-to-haematopoietic transition

Abstract

The endothelial-to-haematopoietic transition (EHT) gives rise to the first murine definitive haematopoietic stem cells (HSCs) in the aorta-gonads-mesonephros (AGM) region around embryonic stage E10.5. While our understanding of the transcriptional networks regulating HSC emergence is ever-increasing, the mechanisms governing the EHT process are still being investigated. The transcription factor Gata3, whose function has previously been characterized in the sympathetic nervous system AGM niche, is expressed directly in the haemogenic endothelium(HE), the tissue giving rise to haematopoietic stem and progenitor cells (HSPCs), prior to EHT. Previous studies have shown that Gata3-expressing endothelial cells (ECs) have a greater capacity to produce haematopoietic colonies, over Gata3-negative EC, while EC-specific Gata3 knockout leads to greatly decreased HSC activity. The cyclin-dependent kinase inhibitorp57Kip2 is notably upregulated in Gata3+ cells of the AGM, indicating a potential role for Gata3 as a cell cycle regulator. This thesis outlines the investigation of Gata3, p57Kip2 and the cell cycle during the EHT, both through functional experiments, as well as via single-cell RNA sequencing (scRNA-seq) of Gata3+ ECs and haematopoietic cells (HCs). ScRNA-seq data of Gata3 knockout (KO) and p57Kip2 KO cells is further used to explore any non-cell cycle related role of Gata3 and p57Kip2 in the HE. This thesis goes on to describe the identification of the potential Gata3 downstream-target CD200R1 and its ligand CD200 as new functional markers for the enrichment of HSPCs, initially discovered within the scRNA-seq dataset via trajectory analysis of Gata3+ ECs differentiating into HSPCs. Finally, this thesis includes the scRNA-seq-level characterization of mesenchymal/stromal cells, a cell type whose AGM niche supportive function is important, but insufficiently defined. Taken together, the data detailed in this thesis will deliver new insights in the generation of the first HSCs and the environment in which they emerge.