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      Subregional localization and characterization of Ly6aGFP-expressing hematopoietic cells in the mouse embryonic head

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          Abstract

          Hematopoietic cell generation in the midgestation mouse embryo occurs through the natural transdifferentiation of temporally and spatially restricted set of hemogenic endothelial cells. These cells take on hematopoietic fate in the aorta, vitelline and umbilical arteries and appear as hematopoietic cell clusters that emerge from the vascular wall. Genetic and live imaging data have supported this. Recently, the embryonic head has been shown to contain fully functional hematopoietic stem cells (HSC). By lineage tracing, cerebrovascular specific endothelial cells were shown to contribute to the postnatal mouse hematopoietic system. Since Ly6aGFP is a marker of all HSCs, some hematopoietic cluster cells and hemogenic endothelial cells in the midgestation mouse aorta, we examine here whether embryonic head HSCs and vascular endothelial cells are positive for this marker. Whereas some head vasculature, single hematopoietic cells and all HSCs are Ly6aGFP expressing, we do not find clusters of hematopoietic cells emerging from the cerebrovasculature that are characteristic of endothelial-to-hematopoietic transition.

          Highlights

          • All midgestation mouse head HSCs and some HPCs are Ly6aGFP expressing.

          • Ly6aGFP is expressed in the major head vasculature, particularly in the hindbrain.

          • No hematopoietic cell clusters are found within the lumen of the head vasculature.

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          Most cited references25

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          Hematopoietic stem cells derive directly from aortic endothelium during development

          A major goal of regenerative medicine is to instruct formation of multipotent, tissue-specific stem cells from induced pluripotent stem cells (iPSCs) for cell replacement therapies. Generation of hematopoietic stem cells (HSCs) from iPSCs or embryonic stem cells (ESCs) is not currently possible, however, necessitating a better understanding of how HSCs normally arise during embryonic development. We previously showed that hematopoiesis occurs through four distinct waves during zebrafish development, with HSCs arising in the final wave in close association with the dorsal aorta. Recent reports have suggested that murine HSCs derive from hemogenic endothelial cells (ECs) lining the aortic floor1,2. Additional in vitro studies have similarly suggested that the hematopoietic progeny of ESCs arise through intermediates with endothelial potential3,4. In this report, we have utilized the unique strengths of the zebrafish embryo to image directly the birth of HSCs from the ventral wall of the dorsal aorta. Utilizing combinations of fluorescent reporter transgenes, confocal timelapse microscopy and flow cytometry, we have identified and isolated the stepwise intermediates as aortic hemogenic endothelium transitions to nascent HSCs. Finally, using a permanent lineage tracing strategy, we demonstrate that the HSCs generated from hemogenic endothelium are the lineal founders of the adult hematopoietic system.
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            In vivo imaging of haematopoietic cells emerging from the mouse aortic endothelium.

            Haematopoietic stem cells (HSCs), responsible for blood production in the adult mouse, are first detected in the dorsal aorta starting at embryonic day 10.5 (E10.5). Immunohistological analysis of fixed embryo sections has revealed the presence of haematopoietic cell clusters attached to the aortic endothelium where HSCs might localize. The origin of HSCs has long been controversial and several candidates of the direct HSC precursors have been proposed (for review see ref. 7), including a specialized endothelial cell population with a haemogenic potential. Such cells have been described both in vitro in the embryonic stem cell (ESC) culture system and retrospectively in vivo by endothelial lineage tracing and conditional deletion experiments. Whether the transition from haemogenic endothelium to HSC actually occurs in the mouse embryonic aorta is still unclear and requires direct and real-time in vivo observation. To address this issue we used time-lapse confocal imaging and a new dissection procedure to visualize the deeply located aorta. Here we show the dynamic de novo emergence of phenotypically defined HSCs (Sca1(+), c-kit(+), CD41(+)) directly from ventral aortic haemogenic endothelial cells.
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              Blood stem cells emerge from aortic endothelium by a novel type of cell transition.

              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.
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                Author and article information

                Contributors
                Journal
                Dev Biol
                Dev. Biol
                Developmental Biology
                Elsevier
                0012-1606
                1095-564X
                01 August 2016
                01 August 2016
                : 416
                : 1
                : 34-41
                Affiliations
                [a ]University of Edinburgh, Centre for Inflammation Research, Queens Medical Research Institute, Edinburgh, UK
                [b ]Erasmus MC Stem Cell Institute, Departments of Cell Biology and Genetics, Erasmus Medical Center, Rotterdam, The Netherlands
                Author notes
                [* ]Corresponding author at: University of Edinburgh, Centre for Inflammation Research, Queens Medical Research Institute, 47 Little France Crescent, Edinburgh EH16 4TJ, UK.University of Edinburgh, Centre for Inflammation Research, Queens Medical Research Institute47 Little France CrescentEdinburghEH16 4TJUK Elaine.Dzierzak@ 123456ed.ac.uk
                Article
                S0012-1606(16)30007-0
                10.1016/j.ydbio.2016.05.031
                4968554
                27235813
                49e55002-1952-433e-b19f-77e51f486443
                © 2016 The Authors

                This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

                History
                : 5 January 2016
                : 7 May 2016
                : 24 May 2016
                Categories
                Short Communication

                Developmental biology
                hematopoietic stem cells,embryonic head,ly6agfp,eht,hematopoietic clusters,hemogenic endothelium

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