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Multipotent haematopoietic stem cells (HSCs) originate in the dorsal aorta (DA) during vertebrate embryogenesis, and after migrating to a permanent niche, give rise to a continuous supply of mature blood cells of all lineages throughout adult life. Previous cell tracing experiments have shown that the cells of the DA migrate here from an early collection of haemangioblasts (bipotential precursors of blood and endothelial cells) which reside in the dorsolateral plate (DLP) mesoderm. Development of HSCs is tightly regulated by a number of key signalling pathways in both the DLP and the DA. In particular, notch signalling is considered an important factor in vascular, arterial and HSC development. Here, the relatively slow development and the spatial separation of definitive haematopoiesis from primitive haematopoiesis in Xenopus laevis has been exploited to reveal the first defect of reduced notch signalling in the Xenopus DA. Two notch inputs to HSC programming have been identified in Xenopus: notch4 and its target genes, esr7 and esr10, are expressed from stage 31, immediately after migrating haemangioblast cells reach the midline of the embryo to form the DA, whilst notch1 is expressed slightly later, from stage 34, and controls expression of two further notch target genes, esr1 and hesr1. Using both morpholino knockdown of these six genes, and chemical inhibition of notch signalling using a specific γ-secretase inhibitor, notch signalling has been demonstrated to be essential for HSC programming but dispensable for earlier haemangioblast and arterial programming. Furthermore, esr1, downstream of both notch1 and notch4, is shown to be responsible for repression of endothelial genes in the DA. Taken together, this demonstrates that a cascade of notch and notch effector genes are essential for the programming of Xenopus HSCs.

The alternative road map describes the separation of lympho-myeloid and myeloid-megakaryocyte-erythroid (myeloid-Mk-E) lineages as the earliest haematopoietic commitment event. However, a number of aspects of this lineage restriction process remain poorly understood. Herein this work identified a lympho-myeloid restricted progenitor in the embryo, which resembles the adult LMPP, and demonstrated that lymphoid lineage restriction is initiated prior to definitive haematopoiesis, much earlier than previously appreciated. In vivo fate mapping showed that lympho-myeloid progenitors significantly contribute to steady state myelopoiesis in the embryo. The early thymic progenitor (ETP) as most primitive cell in the thymus was characterised and demonstrated to sustain B, T and myeloid but not Mk potentials at the single cell level. The ETP therefore largely resembles the cellular properties of lympho-myeloid progenitors in bone marrow and foetal liver, which points to these cells as candidate thymus seeding progenitors (TSP). Furthermore the existence of a putative Mk progenitor was explored within the LSKCD150⁺CD48⁺Gata1^pos compartment of a Gata1 reporter mouse providing the basis for a future prospective characterisation. Finally, this work evaluated the earliest lineage restriction of von Willebrand factor (Vwf)-EGFP⁺ and EGFP^- haematopoietic stem cells (HSCs) through in vitro paired daughter fate mapping. Single Vwf⁺ HSCs showed heterogeneous Mk priming and more frequently sustained Mk potential after cell division. Moreover, analysis of lineage priming between daughter cells revealed the asymmetric expression of key lineage determinants and stem cell regulators, which might be employed as reporters for future fate mapping studies.