Using single cell transcriptomics to characterise endothelial populations in embryonic haematopoiesis

  • Morgan Ann Elizabeth Oatley

    Student thesis: Doctoral ThesisDoctor of Philosophy

    Abstract

    Haematopoietic stem cells (HSCs) possess the unique ability to reconstitute an animal’s entire haematopoietic system. As such, it is inherently interesting to the fields of regenerative medicine and developmental biology to understand how these cells first form in the embryo. Haematopoietic stem and progenitor cells (HSPCs) are understood to derive from a specialised endothelial precursor termed haemogenic endothelium during a narrow window of development. This project was undertaken to better characterise endothelial populations in embryonic haematopoietic tissues in an effort to improve our understanding of the endothelial to haematopoietic transition (EHT). To this end we employed single cell technology, immunofluorescence, flow cytometry and sorting, ex vivo co-culturing assays and a mouse embryonic stem cell (mESC) differentiation system. This work identified two hyaluronan receptors, Cd44 and Stabilin-2 (Stab2) on the surface of endothelial cells derived from the aorta gonad mesonephros (AGM) and yolk sac, respectively. We showed that Cd44 could be used to mark all stages of haematopoietic development arising from the AGM. This enabled us to provide transcriptional data on haemogenic endothelium, identifying potential new regulators of embryonic haematopoiesis and characterising the endothelial precursors as quiescent in nature. Furthermore, ex vivo and in vitro culturing of CD44+ cells identified a functional role for CD44 in the process of EHT. The precise mechanism by which CD44 and its ligand hyaluronan assist in the emergence of HSPCs remains unclear. We further identified the cell surface receptor Stab2 as a distinguishing feature of yolk sac vascular endothelium compared with vascular endothelium of the AGM. Our analysis suggests that EHT is more likely to derive from yolk sac endothelium that is negative for Stab2 expression and by excluding the expression of Stab2 we can enrich for AGM-like haematopoietic progenitors. Our RNA sequencing data suggests that Stab2+ endothelial cells have a transcriptional profile similar to liver sinusoidal endothelial cells, where HSPCs migrate after emergence. This poses an interesting question of whether endothelial niche cells attract haematopoietic progenitors to the yolk sac and their role there. Overall, these two hyaluronan receptors can be used to define distinct populations in the yolk sac and AGM and likely have importance for the emergence of HSPCs.
    Date of Award2019
    Original languageEnglish
    SupervisorInke Nathke (Supervisor) & Christophe Lancrin (Supervisor)

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