Piezo1 is essential for endothelial-to-hematopoietic transition and hematopoietic stem/progenitor cell generation during embryonic development

Abstract Introduction Hematopoietic stem cells (HSCs) form the basis of the hematopoietic system, with essential functions including long-term self-renewal and the generation of all mature blood cell lineages in vivo. Their development relies on the endothelial-to-hematopoietic transition (EHT) of endothelial cells in the aorta-gonad-mesonephros (AGM) region. Mechanical forces, particularly fluid shear stress, have been shown to play a crucial role in HSCs emergence during embryogenesis. Piezo1, a key mechanosensitive ion channel protein in endothelial cells, is known to regulate various physiological processes; however, its specific role in EHT remains unclear. This study aims to elucidate the function of Piezo1 in HSCs generation during EHT, investigate the in vitro hematopoietic function and in vivo transplantation reconstitution potential of hemogenic endothelial cells (HECs) after Piezo1 knockout, and explore the mechanisms by which Piezo1 responds to embryonic hemodynamic changes to regulate EHT in the murine AGM region. Methods We generated an endothelial cell-specific Piezo1 knockout mouse model. Flow cytometry was used to detect fetal liver hematopoietic activity from E12.5 to E14.5 mouse embryos. Meanwhile, the AGM region of E10.5 mice was dissected to quantify the number and proportion of hemogenic endothelial cells and hematopoietic progenitor cells, and the colony-forming ability of AGM-derived cells was further evaluated. Subsequently, we sorted HECs from the AGM region using flow cytometry for in vitro colony-forming assays and OP9 co-culture experiments to examine the impact of Piezo1 knockout on their hematopoietic potential. Concurrently, in vivo transplantation experiments were conducted using mouse AGM-derived cells, and donor chimerism was measured at different time points post-transplantation to evaluate the effect of Piezo1 knockout on hematopoietic reconstitution capacity of HECs. To explore the underlying mechanisms, we performed RNA sequencing (RNA-seq) on sorted HECs from Piezo1 knockout mice, followed by validation of differentially regulated pathways identified by sequencing and conducted rescue experiments using in vitro assays such as OP-9 co-culture. Results Using an endothelial cell-specific Piezo1 knockout mouse model, we found that Piezo1 deletion inhibited fetal liver hematopoietic activity in E12.5 to E14.5 mouse embryos and reduced the proportions of HSCs and hematopoietic stem/progenitor cells (HSPCs) in the fetal liver. We revealed that these effects were not caused by vascular developmental abnormalities in the fetal liver induced by Piezo1 knockout. Subsequently, analysis of the E10.5 AGM region further revealed that Piezo1 knockout decreased the numbers of HECs and hematopoietic progenitor cells, and significantly impaired the colony-forming capacity of AGM-derived cells in vitro. Flow cytometry–based isolation of HECs from the AGM region, followed by colony-forming assays and OP9 co-culture, confirmed that Piezo1 deficiency compromises the in vitro hematopoietic potential of HECs. Moreover, transplantation experiments showed that AGM-derived cells from Piezo1-deficient mice exhibited a reduced chimerism rate after transplantation, indicating impaired hematopoietic reconstitution capacity. Mechanistically, RNA-seq analysis of isolated HECs from Piezo1 knockout mice revealed downregulation of multiple pathways associated with extracellular matrix remodeling and intercellular junctions. Among these, the Rap1–PI3K signaling pathway was significantly suppressed in Piezo1-deficient HECs, and activation of Rap1 partially rescued the in vitro hematopoietic capacity of HECs. Conclusions Based on these findings, we conclude that Piezo1-mediated mechanical effects act as a key factor in the EHT process, influencing the emergence of functional HSCs during mouse embryogenesis. This study elucidates the specific role and underlying mechanism of Piezo1 in EHT regulation, providing new insights for future understanding of the mechanical mechanism of EHT in embryonic hematopoietic development.