Notch ligand Dll4 impairs cell recruitment to aortic clusters and limits blood stem cell generation

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Abstract

<p id="d2080928e446">Hematopoietic stem cells (HSCs) develop from the hemogenic endothelium in cluster structures that protrude into the embryonic aortic lumen. Although much is known about the molecular characteristics of the developing hematopoietic cells, we lack a complete understanding of their origin and the three‐dimensional organization of the niche. Here, we use advanced live imaging techniques of organotypic slice cultures, clonal analysis, and mathematical modeling to show the two‐step process of intra‐aortic hematopoietic cluster (IACH) formation. First, a hemogenic progenitor buds up from the endothelium and undergoes division forming the monoclonal core of the IAHC. Next, surrounding hemogenic cells are recruited into the IAHC, increasing their size and heterogeneity. We identified the Notch ligand Dll4 as a negative regulator of the recruitment phase of IAHC. Blocking of Dll4 promotes the entrance of new hemogenic Gfi1 <sup>+</sup> cells into the IAHC and increases the number of cells that acquire HSC activity. Mathematical modeling based on our data provides estimation of the cluster lifetime and the average recruitment time of hemogenic cells to the cluster under physiologic and Dll4‐inhibited conditions. </p><p class="first" id="d2080928e452">Specification of hematopoietic fate and intra‐aortic cell clusters in the mouse embryo is controlled by Dll4. <div class="boxed-text panel" id="embj2019104270-blkfxd-0002"> <a class="named-anchor" id="embj2019104270-blkfxd-0002">  </a> <div class="figure-container so-text-align-c"> <img alt="" class="figure" src="/document_file/46b0818e-6b55-4e32-9ed7-b17603947aad/PubMedCentral/image/EMBJ-39-e104270-g015.jpg"/> </div> <div class="panel-content"/> </div> </p>

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Most cited references 24

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Accounting for technical noise in single-cell RNA-seq experiments.

Philip Brennecke, Simon Anders, Jong Kim … (2013)

Single-cell RNA-seq can yield valuable insights about the variability within a population of seemingly homogeneous cells. We developed a quantitative statistical method to distinguish true biological variability from the high levels of technical noise in single-cell experiments. Our approach quantifies the statistical significance of observed cell-to-cell variability in expression strength on a gene-by-gene basis. We validate our approach using two independent data sets from Arabidopsis thaliana and Mus musculus.

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Hematopoietic stem cells derive directly from aortic endothelium during development

Julien Y. Bertrand, Neil Chi, Buyung Santoso … (2010)

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.

Jean-Charles Boisset, Wiggert A. van Cappellen, Charlotte Andrieu-Soler … (2010)

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.