| Summary: | <i>De novo</i> generation and physiological expansion of definitive haematopoietic stem cells (dHSCs) occurs exclusively during embryogenesis and is initiated within the dorsal aorta/para-aortic mesenchyme (Ao) of the E11.5 aorta-gonad-mesonephros (AGM) region. The elucidation of the <i>in vivo</i> induction of dHSC emergence and regulation of expansion is likely to be a preparation to the success of generating dHSC <i>in vitro</i> and facilitating an effective and robust expansion of pre-existing dHSCs, thus providing a basis for potentially useful therapeutic translation. Utilising flow cytometry and the <i>in vivo</i> long-term haematopoietic reconstitution assays, we describe the endothelial affiliation of early dHSC as they emerge within the embryonic AGM region and yolk sac, fundamentally defined by a VE-cadherin <sup>+</sup>CD45<sup>+</sup> immunophenotype, and the successive restriction to a haematopoietic identity upon hepatic colonisation. Well-defined haematopoietic colony forming unit-culture (CFU-C) and endothelial network forming assays were used to reveal that the early strong endothelio-haematopoietic promiscuity observed in the early embryo is lost in favour of a mutually exclusive lineage commitment. Experiments using a novel liquid suspension culture system reveal that successful <i>in vitro</i> myeloid differentiation of VE-cadherin <sup>+</sup>CD45<sup>+</sup>dHSCs/multipotent haematopoietic progenitors from the E11.5 AGM region is dependent on an interaction with the endothelial compartment. To directly test the predominating hypothesis of a dorso-ventral polarity in haematopoietic activity of the AGM region I have bisected the Ao from the E11.5 AGM region along the dorso-ventral axis and subjected the resultant ventral (AoV) and dorsal (AoD) aspects to <i>in vivo </i>and <i>in vitro</i> tests. Here I provide the first functional data to support the hypothesis of an anatomical polarity of dHSC distribution. Furthermore, I provide evidence to support an hypothesis that <i>in vivo</i> components of the E10.5-11.5 Ao only permit ventral emergence and expansion of dHSCs. Investigation into the transcriptional activity of the E11.5 AoV and AoD highlight the differential expression of GATA2, GATA3, HoxB4, Runxl, BMP4, Noggin and Chordin. These data demonstrate the conductive nature of the AGM region, which after structural disruption not only supports the <i>ex vivo</i> maintenance of dHSC activity but also exhibits a marked capacity for rapid stem cell expansion. Thus, in our hands we have a powerful system with the potential to allow useful investigation into the fundamental processes that occupy the field of stem cell biology; the mechanisms of stem cell emergence, induction and expansion.
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