LAUSR

Insight into the role of the niche in which the first haematopoietic stem cells (HSCs) emerge in vivo is still lacking. Cells expressing NG2, a cell surface proteoglycan found in both vasculogenic and angiogenic vasculature, have been shown to support adult HSC maintenance. Whether NG2+ cells also control HSC generation in the mouse embryo is still unknown. NG2 expression in the aortic mural cells starts shortly before HSC generation and some NG2+ cells also express Runx1, a key haematopoietic transcription factor required for HSC emergence. We deleted Runx1 in NG2 expressing cells by crossing Runx1fl/fl mice with NG2-Cre mice. In vitro and in vivo functional assays were performed in E10 and E11 mouse embryos to assess haematopoietic activity. 3D confocal imaging of the intact aorta was further used to confirm our results. To investigate whether NG2+ and NG2+Runx1+ cells are haematopoietic and/or haematopoietic precursors, cell sorting and colony-forming assays were carried out on WT, Runx1 GFP and NG2Cre-TdTomato transgenic mice. 3D confocal imaging showed that intra-aortic haematopoietic clusters are dramatically reduced in number in homozygous KO mouse embryos compared to WT controls. This was further confirmed by functional in vitro and in vivo assays. Both haematopoietic progenitors and haematopoietic stem cell activities are affected in the KO AGM. Depletion of Runx1 in NG2+ cells drastically impairs all haematopoietic progenitor numbers in the AGM of both E10.5 and E11 mouse embryos and, in vivo, KO AGM had a reduced capacity of reconstituting adult irradiated recipient mice. NG2+ cells (or NG2+Runx1+) are not haematopoietic nor haematopoietic precursors suggesting that NG2+Runx1+ cells act as a supportive niche for haematopoietic progenitor and stem cell generation at this stages. Ongoing experiments aim to test whether this is time- and organ-dependent.