LACK OF BLOOD FLOW BLOCKS MOUSE HSC DEVELOPMENT AT THE PRO-HSC STAGE

Abstract

Hematopoietic stem cells (HSCs) emerge from aortic hemogenic endothelium through a continuum of precursors, the pro- and pre-HSCs. Studies in zebrafish have implicated blood flow in this process. In the mouse, embryos without blood flow die before transplantable HSCs appear, precluding a direct assessment of the role of flow in HSC emergence. With the recent identification of pro-HSCs that emerge early in development and can be matured ex vivo into functional HSCs, it is now possible to address this question. Here, we assessed the specification and development of the HSC lineage in Ncx1-/- embryos lacking a heartbeat and circulation. We found that hemogenic endothelium and pro-HSCs emerge at a normal frequency, although in reduced numbers. However, Ncx1-/- pro-HSCs were severely impaired in their function and did not give rise to functional HSCs ex vivo. These data indicate that although the first steps of HSC lineage development take place in the absence of circulation, HSC precursors fail to complete their maturation, possibly due to the absence of key signaling events triggered by blood flow. Single cell gene expression profiling showed an aberrant signature in pro-HSCs and hemogenic endothelium isolated from Ncx1-/- embryos, with evidence of a block in hematopoietic development. Moreover, our data showed a decrease in Jag1-mediated Notch signaling in E9.5 Ncx1-/- hemogenic endothelium and pro-HSC, with a role for Notch signaling in the maturation of pro-HSC into HSCs further supported by functional assays. Finally, we found that lack of blood flow resulted in defects in the microenvironment, with absence of peri-aortic smooth muscle and a decrease of macrophages. Exploring what other flow-related signals affect the maturation of pro-HSC along the HSC lineage will be of interest in light of the future optimizing of in vitro HSC generation for therapeutic purposes.