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      Fli + etsrp + Hemato-Vascular Progenitor Cells Proliferate at the Lateral Plate Mesoderm during Vasculogenesis in Zebrafish

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          Abstract

          Background

          Vasculogenesis, the de novo formation of blood vessels from precursor cells is critical for a developing embryo. However, the signals and events that dictate the formation of primary axial vessels remain poorly understood.

          Methodology/Principal Findings

          In this study, we use ets-related protein-1 ( etsrp), which is essential for vascular development, to analyze the early stages of vasculogenesis in zebrafish. We found etsrp + cells of the head, trunk and tail follow distinct developmental sequences. Using a combination of genetic, molecular and chemical approaches, we demonstrate that fli + etsrp + hemato-vascular progenitors (FEVPs) are proliferating at the lateral plate mesoderm (LPM). The Shh-VEGF-Notch-Hey2 signaling pathway controls the proliferation process, and experimental modulation of single components of this pathway alters etsrp + cell numbers at the LPM.

          Conclusions/Significance

          This study for the first time defines factors controlling proliferation, and cell numbers of pre-migratory FEVPs in zebrafish.

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          Most cited references44

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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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              The vascular anatomy of the developing zebrafish: an atlas of embryonic and early larval development.

              We have used confocal microangiography to examine and describe the vascular anatomy of the developing zebrafish, Danio rerio. This method and the profound optical clarity of zebrafish embryos make it possible to view the entire developing vasculature with unprecedented resolution. A staged series of three-dimensional images of the vascular system were collected beginning shortly after the onset of circulation at 1 day postfertilization through early- to midlarval stages at approximately 7 days postfertilization. Blood vessels in every region of the animal were imaged at each stage, and detailed "wiring patterns" were derived describing the interconnections between every major vessel. We present an overview of these data here in this paper and in an accompanying Web site "The interactive atlas of zebrafish vascular anatomy" online at (http://eclipse.nichd.nih.gov/nichd/lmg/redirect.html). We find a highly dynamic but also highly stereotypic pattern of vascular connections, with different sets of primitive embryonic vessels severing connections and rewiring in new configurations according to a reproducible plan. We also find that despite variation in the details of the vascular anatomy, the basic vascular plan of the developing zebrafish shows strong similarity to that of other vertebrates. This atlas will provide an invaluable foundation for future genetic and experimental studies of vascular development in the zebrafish.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2011
                25 February 2011
                : 6
                : 2
                : e14732
                Affiliations
                [1]Department of Pediatrics, CRI Developmental Vascular Biology Program, Translational and Biomedical Research Center, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America
                Katholieke Universiteit Leuven, Belgium
                Author notes

                Conceived and designed the experiments: CZC GW RR. Performed the experiments: CZC IR MOS GVS KP. Analyzed the data: CZC IR MOS GVS KP GW RR. Contributed reagents/materials/analysis tools: CZC KP GW RR. Wrote the paper: GW RR.

                [¤]

                Current address: Life Sciences, Delhi, India

                Article
                10-PONE-RA-21031R1
                10.1371/journal.pone.0014732
                3045372
                21364913
                0530612c-9df2-4bb9-8566-8422e15208e2
                Chun et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                History
                : 13 July 2010
                : 29 January 2011
                Page count
                Pages: 12
                Categories
                Research Article
                Developmental Biology
                Developmental Biology/Cell Differentiation
                Developmental Biology/Developmental Molecular Mechanisms

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                Uncategorized

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