The endothelial to haematopoietic transition (EHT) is the process whereby haemogenic endothelium differentiates into haematopoietic stem and progenitor cells (HSPCs). The intermediary steps of this process are unclear, in particular the identity of endothelial cells that give rise to HSPCs is unknown. Using single-cell transcriptome analysis and antibody screening, we identify CD44 as a marker of EHT enabling us to isolate robustly the different stages of EHT in the aorta-gonad-mesonephros (AGM) region. This allows us to provide a detailed phenotypical and transcriptional profile of CD44-positive arterial endothelial cells from which HSPCs emerge. They are characterized with high expression of genes related to Notch signalling, TGFbeta/BMP antagonists, a downregulation of genes related to glycolysis and the TCA cycle, and a lower rate of cell cycle. Moreover, we demonstrate that by inhibiting the interaction between CD44 and its ligand hyaluronan, we can block EHT, identifying an additional regulator of HSPC development.

Endothelial cells line all blood vessels, where they coordinate blood vessel formation and the blood-tissue barrier via regulation of cell-cell junctions. The nucleus also regulates endothelial cell behaviors, but it is unclear how the nucleus contributes to endothelial cell activities at the cell periphery. Here, we show that the nuclear-localized linker of the nucleoskeleton and cytoskeleton (LINC) complex protein SUN1 regulates vascular sprouting and endothelial cell-cell junction morphology and function. Loss of murine endothelial Sun1 impaired blood vessel formation and destabilized junctions, angiogenic sprouts formed but retracted in SUN1-depleted sprouts, and zebrafish vessels lacking Sun1b had aberrant junctions and defective cell-cell connections. At the cellular level, SUN1 stabilized endothelial cell-cell junctions, promoted junction function, and regulated contractility. Mechanistically, SUN1 depletion altered cell behaviors via the cytoskeleton without changing transcriptional profiles. Reduced peripheral microtubule density, fewer junction contacts, and increased catastrophes accompanied SUN1 loss, and microtubule depolymerization phenocopied effects on junctions. Depletion of GEF-H1, a microtubule-regulated Rho activator, or the LINC complex protein nesprin-1 rescued defective junctions of SUN1-depleted endothelial cells. Thus, endothelial SUN1 regulates peripheral cell-cell junctions from the nucleus via LINC complex-based microtubule interactions that affect peripheral microtubule dynamics and Rho-regulated contractility, and this long-range regulation is important for proper blood vessel sprouting and junction integrity.

Endothelial cells line all blood vessels and coordinate blood vessel formation and the blood-tissue barrier via endothelial cell-cell junctions. The nucleus also regulates endothelial cell behaviors, but the mechanisms are poorly understood. Here we show that nuclear-localized SUN1, a LINC complex component that connects the nucleus to the cytoskeleton, regulates endothelial cell-cell junction communication and blood vessel formation. Loss of murine endothelial Sun1 impaired blood vessel formation and destabilized junctions. At the cellular level, SUN1 stabilized endothelial cell-cell junctions and promoted barrier function. Abnormal SUN1-depleted junctions resembled those seen with loss of microtubules, and they were accompanied by impaired microtubule dynamics and actomyosin hypercontractility. Angiogenic sprouts formed but retracted in SUN1-depleted endothelial cells, and vessels of zebrafish lacking SUN1 had abnormal extension and were defective in forming connections. Thus, endothelial SUN1 regulates peripheral cell-cell junctions from the nucleus, likely via microtubule-based interactions, and this long-range regulation is important for blood vessel formation and barrier function.

The Yolk Sac (YS) and Aorta-Gonad-Mesonephros (AGM) are two major haematopoietic regions during embryonic development. Interestingly, AGM is the only one generating haematopoietic stem cells (HSCs). To identify the difference between AGM and YS, we compared them using singlecell RNA sequencing between 9.5 and 11.5 days of mouse embryonic development and identified cell populations using CONCLUS, a new computational tool. The AGM was the only one containing neurons and a specific mesenchymal population, while the YS major component was an epithelial population expressing liver marker genes. In addition, the YS contained a major endothelial population expressing Stab2, a hyaluronan receptor, also highly expressed by liver endothelium. We demonstrated that the YS haematopoietic potential was restricted to Stab2negative cells and that ectopic expression of Stab2 could reduce blood cell formation from endothelium. Our results indicate that the AGM is a tissue more favourable to HSCs development than the YS because of its microenvironment and the nature of its endothelial cells.

The endothelial to haematopoietic transition (EHT) is the process whereby haemogenic endothelium differentiates into haematopoietic stem and progenitor cells (HSPCs). The intermediary steps of this process are unclear, in particular the identity of endothelial cells that give rise to HSPCs is unknown. Using single-cell transcriptome analysis and antibody screening we identified CD44 as a new marker of EHT enabling us to isolate robustly the different stages of EHT in the aorta gonad mesonephros (AGM) region. This allowed us to provide a very detailed phenotypical and transcriptional profile for haemogenic endothelial cells, characterising them with high expression of genes related to Notch signalling, TGFbeta/BMP antagonists (Smad6, Smad7 and Bmper) and a downregulation of genes related to glycolysis and the TCA cycle. Moreover, we demonstrated that by inhibiting the interaction between CD44 and its ligand hyaluronan we could block EHT, identifying a new regulator of HSPC development.