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Blood, Vol. 111, Issue 3, 1147-1156, February 1, 2008
Live imaging of emerging hematopoietic stem cells and early thymus colonization
Blood Kissa et al.
111: 1147
Supplemental Materials for: Kissa et al
Files in this Data Supplement:
- Video 1. Xz confocal scanning through the trunk of a 38 hpf CD41-gfp embryo, pre-incubated with the vital red fluorescent counterstain BodipyTR (see Figure 1C) (MOV, 767 KB)
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Depth is indicated in the lower left corner. Annotations have the same meaning as in Figure 1. A yellow arrowhead points at the first of a series of GFPlow cells appearing in the DP joint / AGM along the stack; asterisk, pronephric duct associated non-hematopoietic cell.
- Video 2. Rolling of CD41-GFPlow precursors in the PCV at 54 hpf (MOV, 1,229 KB)
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In this real-time video sequence, at the beginning of the movie, a GFPlow precursor cell is slowly rolling on the ventral wall of the PCV (below is the autofluorescence of the yolk tube) while two GFPhigh prothrombocytes successively circulate quickly past it. At 10 sec., another such GFPlow cell is shown by DIC video-microscopy rolling on the dorsal wall of the PCV, past a similar GFPlow cell stalled on the lateral wall of the PCV. At 17 sec., the sequence switches to showing the low fluorescence of the stalled cell, and the high fluorescence of a prothrombocyte that circulates past it.
- Video 3. Prothrombocytes in the CHT entering the circulation at 3 dpf (MOV, 7,165 KB)
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Spinning-disk confocal time-lapse fluorescence microscopy of a CD41-gfp embryo pre-incubated with BodipyTR (1 confocal plane, imaged every 15 sec.). Here the red fluorescent Bodipy TR mostly fills the extracellular space of the CHT and beneath it the CV lumen.
Among a group of four GFPhigh prothrombocytes, likely sister cells, two are released into the CV circulation during the sequence, linked through a long tether to each other as well as to the remaining two prothrombocytes not yet released. At t=15.5 min., to the left, another circulating (pro)thrombocyte transiently stops, also bound to the CV wall by a tether.
- Video 4. Intravasation of CD41-GFPlow cells from the AGM into the PCV at 54 hpf (MOV, 1,841 KB)
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Time-lapse confocal imaging of GFP (green) and BodipyTR (red) fluorescence, overlaid on transmitted light image (see Figure 3A). Three CD41-GFPlow cells (yellow, blue and red dots) initially in the AGM mesenchyme abluminal to the PCV enter one after another into the PCV lumen and circulation within 3 hrs of follow-up.
- Video 5 (=movie 4-stereo). Intravasation of CD41-GFPlow cells from the AGM into the PCV at 54 hpf (MOV, 3,762 KB)
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To view this stereoscopic version of movie 3, fuse the two images by squinting, wait until stereopsis (3D vision) is attained, then start the movie.
- Video 6. Intravasation of CD41-GFPlow cells from the AGM into the PCV at 48 hpf (MOV, 860 KB)
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Spinning disk confocal fluorescence imaging of a PU.1 Mo-injected embryo. Two CD41-GFPlow cells (probably sister cells) initially in the AGM mesenchyme abluminal to the PCV enter one after another into the PCV lumen.
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