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.

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Blood as connective tissue potentially contains evidence of all processes occurring within the organism, at least in trace amounts (Petricoin et al., 2006) [1]. Because of their small size, peptides penetrate cell membranes and epithelial barriers more freely than proteins. Among the peptides found in blood, there are both fragments of proteins secreted by various tissues and performing their function in plasma and receptor ligands: hormones, cytokines and mediators of cellular response (Anderson et al., 2002) [2]. In addition, in minor amounts, there are peptide disease markers (for example, oncomarkers) and even foreign peptides related to pathogenic organisms and infection agents. To propose an approach for detailed peptidome characterization, we carried out an LC–MS/MS analysis of blood serum and plasma samples taken from 20 healthy donors on a TripleTOF 5600+ mass-spectrometer. We prepared samples based on our previously developed method of peptide desorption from the surface of abundant blood plasma proteins followed by standard chromatographic steps (Ziganshin et al., 2011) [3]. The mass-spectrometry peptidomics data presented in this article have been deposited to the ProteomeXchange Consortium (Deutsch et al., 2017) [4] via the PRIDE partner repository with the dataset identifier PXD008141 and 10.6019/PXD008141.

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.

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.

Erythropoiesis occurs through several waves during embryonic development.Although the source of the primitive wave is well characterized, the origin of erythrocytes later in embryogenesis is less clear due to overlaps between the different erythroid waves. Using the miR144/451-GFP mouse model to track cells expressing the erythroid microRNAs miR144/451, we identified cells co-expressing VE-Cadherin and GFP in the yolk sac between E9.5 and E12. This suggested the existence of hemogenic endothelial cells committed to erythropoiesis (Ery-HEC). We showed that these cells were capable of generating erythrocytes ex vivo and we demonstrated that the formation of Ery-HEC was independent of the Runx1 gene expression. Using transcriptome analysis, we demonstrated that these cells coexpressed endothelial and erythroid genes such as Hbb-bh1 and Gata1 but we were surprised to detect the primitive erythroid genes Aqp3 and Aqp8 suggesting the formation of primitive erythrocytes at a much later time point than initially thought.Finally, we showed that enforced expression of Gata1 in endothelial cells was enough to initiate the erythroid transcriptional program.

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