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Blood stem cells emerge from aortic endothelium by a novel type of cell transition

Abstract

The ontogeny of haematopoietic stem cells (HSCs) during embryonic development is still highly debated, especially their possible lineage relationship to vascular endothelial cells1,2. 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 floor3. But although some authors have presented evidence that HSCs may arise directly from the aortic floor into the dorsal aortic lumen4, others support the notion that HSCs first emerge within the underlying mesenchyme5. 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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Figure 1: Endothelial cells from the aortic floor exit into the sub-aortic space to become haematopoietic cells.
Figure 2: The haematopoietic cells emerging by EHT of aortic floor endothelial cells are HSPCs that seed the successive haematopoietic organs.
Figure 3: EHT events correlate with a phase of DA radial shrinking, preceded by a phase of DA radial expansion.
Figure 4: Runx1 expression is required for a successful EHT.

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Acknowledgements

We thank the Zebrafish International Resource Center at the University of Oregon and L. Zon for providing KDR–GFP and Lmo2–Dsred transgenic zebrafish, respectively, Y. Blum and M. Affolter for the KDR–dTomato plasmid, and C. Herbomel and O. Bihan-Poudec for graphic artwork.

Author Contributions K.K. performed the confocal fluorescence imaging, data analysis, and morpholino or plasmid microinjections; P.H. performed the video-enhanced DIC imaging and wrote the manuscript with input from K.K.

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Correspondence to Philippe Herbomel.

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Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-2 with Legends and Legends for Supplementary Movies 1-6. (PDF 2252 kb)

Supplementary Movie 1

In this movie we see the time-lapse confocal fluorescence imaging of a zebrafish embryo showing the process of exit of aorta floor endothelial cells into the sub-aortic space to become hematopoietic cells - see Supplementary Information file for full Legend. (MOV 4429 kb)

Supplementary Movie 2

This movies shows the process of the emergence of an hematopoietic cell from the aorta floor in longitudinal view, with the transverse view of the same cell shown as Supplementary Movie 3 - see Supplementary Information file for full Legend. (MOV 2276 kb)

Supplementary Movie 3

This movie shows the process of the emergence of an hematopoietic cell from the aorta floor in transverse view, with the longitudinal view of the same cell shown as Supplementary Movie 2 - see Supplementary Information file for full Legend. (MOV 1081 kb)

Supplementary Movie 4

In this movie we see the time-lapse confocal fluorescence imaging of a zebrafish embryo showing hematopoietic cells emerged from the aorta floor entering the microstroma of the axial vein roof and from there the blood circulation - see Supplementary Information file for full Legend. (MOV 3881 kb)

Supplementary Movie 5

In this movie we see the time-lapse confocal fluorescence imaging of a zebrafish embryo then larva from 1 to 4 days post fertilization, showing the correlation between aorta radial expansion then constriction and the period of endothelial to hematopoietic transition (EHT) of aorta floor cells - see Supplementary Information file for full Legend. (MOV 12521 kb)

Supplementary Movie 6

In this movie we see the time-lapse confocal fluorescence imaging of a zebrafish embryo in which runx1 expression was knocked down, showing that only few aorta floor cells initiate EHT and that the process is abortive, ending with the explosive death of the cell - see Supplementary Information file for full Legend. (MOV 5113 kb)

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Kissa, K., Herbomel, P. Blood stem cells emerge from aortic endothelium by a novel type of cell transition. Nature 464, 112–115 (2010). https://doi.org/10.1038/nature08761

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