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Hematopoietic stem and progenitor cells (HSPCs) are multipotent cells giving rise to all blood lineages during life. HSPCs emerge from the ventral wall of the dorsal aorta (VDA) during a specific timespan in embryonic development through endothelial hematopoietic transition (EHT). We investigated the ontogeny of HSPCs in mutant zebrafish embryos lacking functional pten, an important tumor suppressor with a central role in cell signaling. Through in vivo live imaging, we discovered that in pten mutant embryos a proportion of the HSPCs died upon emergence from the VDA, an effect rescued by inhibition of phosphatidylinositol-3 kinase (PI3K). Surprisingly, inhibition of PI3K in wild-type embryos also induced HSPC death. Surviving HSPCs colonized the caudal hematopoietic tissue (CHT) normally and committed to all blood lineages. Single-cell RNA sequencing indicated that inhibition of PI3K enhanced survival of multipotent progenitors, whereas the number of HSPCs with more stem-like properties was reduced. At the end of the definitive wave, loss of Pten caused a shift to more restricted progenitors at the expense of HSPCs. We conclude that PI3K signaling tightly controls HSPCs survival and both up- and downregulation of PI3K signaling reduces stemness of HSPCs.

In vertebrates, haematopoietic stem/progenitor cells (HSPCs) first emerge in the aorta-gonad-mesonephros (AGM) before colonizing transitory and subsequently definitive haematopoietic organs allowing haematopoiesis throughout adult life. Here we identify an unexpected primitive macrophage population accumulated in the dorsal mesenteric mesoderm surrounding the dorsal aorta of the human embryo and study its function in the transparent zebrafish embryo. Our study reveals dynamic interactions occurring between the HSPCs and primitive macrophages in the AGM. Specific chemical and inducible genetic depletion of macrophages or inhibition of matrix metalloproteinases (Mmps) leads to an accumulation of HSPCs in the AGM and a decrease in the colonization of haematopoietic organs. Finally, in vivo zymography demonstrates the function of primitive macrophages in extracellular matrix degradation, which allows HSPC migration through the AGM stroma, their intravasation, leading to the colonization of haematopoietic organs and the establishment of definitive haematopoiesis.

During embryogenesis of all vertebrates, haematopoietic stem/progenitor cells (HSPCs) extrude from the aorta by a complex process named endothelial-to-haematopoietic transition (EHT). HSPCs will then colonize haematopoietic organs allowing haematopoiesis throughout adult life. The mechanism underlying EHT including the role of each aortic endothelial cell (EC) within the global aorta dynamics remains unknown. In the present study, we show for the first time that EHT involves the remodelling of individual cells within a collective migration of ECs which is tightly orchestrated, resulting in HSPCs extrusion in the sub-aortic space without compromising aorta integrity. By performing a cross-disciplinary study which combines high-resolution 4D imaging and theoretical analysis based on the concepts of classical mechanics, we propose that this complex developmental process is dependent on mechanical instabilities of the aorta preparing and facilitating the extrusion of HSPCs. [ABSTRACT FROM AUTHOR]

The ontogeny of haematopoietic stem cells (HSCs) during embryonic development is still highly debated, especially their possible lineage relationship to vascular endothelial cells. The first anatomical site from which cells with long-term HSC potential have been isolated is the aorta-gonad-mesonephros (AGM), more specifically the vicinity of the dorsal aortic floor. But although some authors have presented evidence that HSCs may arise directly from the aortic floor into the dorsal aortic lumen, others support the notion that HSCs first emerge within the underlying mesenchyme. Here we show by non-invasive, high-resolution imaging of live zebrafish embryos, that HSCs emerge directly from the aortic floor, through a stereotyped process that does not involve cell division but a strong bending then egress of single endothelial cells from the aortic ventral wall into the sub-aortic space, and their concomitant transformation into haematopoietic cells. The process is polarized not only in the dorso-ventral but also in the rostro-caudal versus medio-lateral direction, and depends on Runx1 expression: in Runx1-deficient embryos, the exit events are initially similar, but much rarer, and abort into violent death of the exiting cell. These results demonstrate that the aortic floor is haemogenic and that HSCs emerge from it into the sub-aortic space, not by asymmetric cell division but through a new type of cell behaviour, which we call an endothelial haematopoietic transition. [ABSTRACT FROM AUTHOR]

Summary: Although the ontogeny of hematopoietic stem cells (HSCs) in vertebrates has been studied intensely, a lineage relationship between the HSCs found in the developmentally successive hematopoietic organs remains to be shown. By using an in situ photoactivatable cell tracer in the transparent zebrafish embryo, we demonstrated that definitive blood precursors appeared between the dorsal aorta and axial vein, validating the homology of this tissue with the AGM (aorta-gonad-mesonephros) of amniotes. These cells first migrated through the blood to a previously undescribed caudal hematopoietic tissue (CHT), where they differentiated, expanded, and further migrated to seed the definitive hematopoietic organs, the thymus and kidney. Immigrants on the way to the thymus expressed c-myb and ikaros but not rag1; they were probably no longer HSCs, however, because they lacked scl and runx1 expression, unlike immigrants to the kidney. The CHT thus has a hematopoietic function similar to that of the mammalian fetal liver. [Copyright &y& Elsevier]