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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)1,2,3. Immunohistological analysis of fixed embryo sections has revealed the presence of haematopoietic cell clusters attached to the aortic endothelium where HSCs might localize4,5,6. 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 system8,9 and retrospectively in vivo by endothelial lineage tracing5,10 and conditional deletion experiments11. 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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Figure 1: Visualization of intra-aortic haematopoietic cells by confocal imaging on non-fixed E10 embryo slices.
Figure 2: Emergence of phenotypically defined HSCs on embryo slices.
Figure 3: Emergence of phenotypically defined HSCs in whole E10 Ly-6A-GFP embryos.


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We thank F. Grosveld for critical discussions, T. Graf and T. Schroeder for the CD41–YFP mice and the department of Neuroscience (Erasmus MC) for the tissue chopper. This work was supported by NWO (Vidi) grant 917-76-345 (C.R., J.-C.B.), NIH R37 DKO54077 (E.D.) and BSIK SCDD 03038 (E.D.).

Author Contributions C.R. and E.D. conceived ideas. C.R. designed the research. C.R and J.-C.B. performed the experiments, analysed the data, interpreted the experiments and made the figures. C.R. and C.A.S. developed the embryo slicing technique. W.v.C. assisted with the confocal microscopy experiments. C.R., J.-C.B. and N.G. made the 3D movies. C.R. and W.v.C. made the 2D movies. C.R., J.-C.B., N.G. and E.D. wrote the manuscript. All authors discussed the results and commented on the manuscript.

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Correspondence to Catherine Robin.

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Supplementary information

Supplementary Figures

This file contains Supplementary Figures 1-3 with Legends. (PDF 904 kb)

Supplementary Movie 1

This movie shows the 3D reconstruction of the aorta from a E10 embryo slice. Ventral and dorsal sides of the aorta are specified. The boxed area shows a CD31+ ventral aortic cluster (red, CD31). (MOV 6034 kb)

Supplementary Movie 2

This movie shows the emergence of a Ly-6A GFP+ cell (white arrow) directly from the CD31+ ventral aortic endothelium (Example 1, 20x lens; green, Ly-6A GFP; red, CD31). (MOV 4001 kb)

Supplementary Movie 3

This movie shows the emergence of a Ly-6A GFP+ cell (white arrow) directly from the CD31+ ventral aortic endothelium (Example 2, 20x lens; green, Ly-6A GFP; red, CD31). (MOV 5345 kb)

Supplementary Movie 4

The first part of this movie shows the 3D reconstruction of the aorta from a E10 Ly-6A GFP embryo slice. Ventral/dorsal and front/back sides of the aorta are specified. The white box represents the area where two Ly-6A GFP+ cells will emerge in position of the white and yellow arrows. The second part shows the white box area from the front side (left) and back side (right) perspective of the aorta (40x lens; green, Ly-6A GFP; red, CD31). (MOV 5000 kb)

Supplementary Movie 5

This movie shows the emergence of a Ly-6A GFP+ cell (shown in Supplementary Movie 4, white arrow) in 2D (one z-stack) after deconvolution (40x lens; green, Ly-6A GFP; red, CD31). (MOV 4263 kb)

Supplementary Movie 6

This movie shows the emergence of a Ly-6A GFP+ cell (white arrow) directly from the CD31+ aortic endothelium (40x lens; green, Ly-6A GFP; red, CD31). Ventral and dorsal sides of the aorta are specified. (MOV 9939 kb)

Supplementary Movie 7

This movie shows the emergence of CD41-YFP+ cells (yellow arrow) directly from the ventral CD41- aortic endothelium (40x lens; yellow, CD41-YFP). The emergence and the concomitant YFP expression of the newly formed cells are shown in transmitted light (on the left) and fluorescence (on the right). (MOV 6450 kb)

Supplementary Movie 8

This movie shows the 3D reconstruction of the ventral and lateral sides of an aorta portion from a whole E10 embryo. Notice the hematopoietic clusters attached to the endothelium (40x lens; green, Ly-6A GFP; red, CD31). (MOV 5553 kb)

Supplementary Movie 9

This movie shows the emergence of a GFP+ cell from the aortic endothelium (20x lens; green, Ly-6A GFP). (MOV 7817 kb)

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Boisset, JC., van Cappellen, W., Andrieu-Soler, C. et al. In vivo imaging of haematopoietic cells emerging from the mouse aortic endothelium. Nature 464, 116–120 (2010).

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