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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:
- Table S1. In vivo measurements of florescence intensities of GFPlow HSC-like precursors and GFPhigh prothrombocytes in CD41-gfp transgenic embryos (PDF, 16 KB) -
Confocal image stacks were generated in constant conditions (under the 40× oil objective of a Leica SPE, every 3 µm in z, at 30% laser illumination), and the florescence level of each cell was measured in the confocal plane in which it was maximum, using the Leica SPE software.
Values (mean and standard deviation) are given in arbitrary units. From 3 dpf onwards, the large number and very high florescence of GFPhigh cells in the CHT and caudal AGM precluded a reliable in situ measurement of the florescence level of GFPlow cells.
- Figure S1. Cell divisions of CD41-GFPlow precursors in the AGM, CHT, thymus and kidney (PDF, 928 KB) -
Lateral views, rostral to the left.
(A) Mitosis of one among two CD41-GFPlow cells in the DP joint (AGM) at 48 hpf. Asterisk, non hematopoietic, pronephric duct (pd) associated cell.
(B) Mitosis of a CD41-GFPlow early precursor in the CHT at 38 hpf.
(C) Confocal fluorescence colocalization analysis following anti-GFP (green) and anti-phosphohistone H3 (red) immunohistochemistry at 5.5 dpf, in a single confocal plane. Both thymus and kidney images (lateral views, from the same embryo) show the overlay of DIC, red and green fluorescence; for each image a histogram of the three signal intensities along the red line is given below. The left one establishes the presence in the thymus along the red line of 1 mitotic GFP− cell and 3 GFP+ cells, one of which is mitotic (red+green). The histogram of intensities along the red line in the kidney image reveals one GFPhigh prothrombocyte and two GFPlow cells, one of which in mitosis (red+green).
ntc, notochord; m, thymus-proximal muscle; t, thymus; ba, branchial arches (gills); g, pronephric glomerulus.
- Figure S2. Mitosis of a CD41-GFPlow hematopoietic precursor in the circulation (PDF, 165 KB) -
DIC video-microscopy at 48 hpf in the yolk sac circulation valley (duct of Cuvier, see Figure 1A), where the blood flows directly over the yolk surface. Time points are indicated. At t=0, Cell 1 is already in anaphase. Three other CD41-GFPlow cells of similar morphology and fluorescence intensity (numbered 2 to 4) successively roll by it, and sometimes stall in contact with it, during the sequence. A macrophage (M) present at t=0 is seen at t=8 min adhering to the dividing Cell 1 through a 30 µm long tether (white arrows), by which it is pulling one of the two daughter cells to be (better seen on the video sequence). A granulocyte (g) passes by in the blood flow at t=9.5min. Full video sequence available upon request.
- Figure S3. Fate of CD41-GFPlow AGM cells labelled at 48 hpf by uncaging of caged rhodamine-dextran (Supplement to Figure 2) (PDF, 706 KB) -
(A) Rhodamine uncaging in a GFP+ cell cluster at the caudal end of the AGM (above the uro-genital opening) led 65 hpf later, to (B) several labelled cells further caudally in the CHT (arrows and asterisks) and (C) three labelled cells in the thymus, and by 5.5 dpf to (D) many labelled cells in the thymus. All left lateral views, rostral to the left. UGO, uro-genital opening. Scale bars, 20 µm.
- Figure S4. CD41−GFP+ hematopoietic cells are suppressed in runx1 morphants and DAPT-treated embryos (PDF, 612 KB) -
Lateral views, rostral to the left.
(A-H, K, L) Green fluorescence overlaid on DIC image; (I,J) DIC only; (M-P) Fluorescence only.
(A-D) At 54hpf, many CD41-GFP+ cells are observed all along the DA-PCV joint (A) and CHT (C) of control embryos, but not in runx1-morpholino injected morphants (B,D).
(E-H) At 75hpf, the DA-PCV joint has become thicker in control embryos, and colonized by numerous CD41-GFP+ cells (E). In runx1 morphants, the DA and PCV remain closely juxtaposed (F,arrow), with no GFP+ cells in between; note that the non hematopoietic GFP+ cells associated with the pronephric duct are still present (F). The CHT in control embryos is seeded by many CD41-GFP+ cells (G), while very few cells are found in the CHT in runx1 morphants (H). (I-J) Accumulated thymocytes are visible at 72 hpf by DIC in control embryos (I), but not in runx1 morphants (J); fluorescence images confirm this (K,L).
(M-P) CD41-gfp embryos incubated with DAPT since the onset of gastrulation (shield stage) have very few GFP+ cells in the trunk and CHT at 54 and 72 hpf (N,P) compared to control embryos (M,O). Scale bar (A-L) 50 µm.
- Figure S5. The CD41-GFPlow blood precursors arise within the c-myb+ cell cord in the DP joint (PDF, 108 KB) -
Whole-mount in situ hybridization performed on 36 hpf embryos; all lateral views, rostral to the left.
(A,B) Alkaline phosphatase-based detection of CD41-gfp (A) and c-myb (B) transcripts in the trunk; c-myb+ cells form a quasi-continuous cell cord all along the DP joint, i.e. between DA and PCV; CD41-gfp+ cells are also all along the DP joint but sparser.
(C) Double fluorescent detection of CD41-gfp and c-myb transcripts in embryos siblings of those shown in (A,B). CD41-gfp transcripts are detected with green (FITC) tyramide and c-myb transcripts with red (Cy3) tyramide in (a-c), and in the reverse combination in (d-e). The corresponding DIC/Nomarski image is shown to the right; arrows indicate matching cells in each set of three panels. Scale bars, 50 µm (A,B) and 20 µm (C).
- Figure S6. Dynamics of cell release from AGM to CHT: CD41-gfp expression marks competency to leave the AGM (Supplement to Figure 5) (PDF, 152 KB) -
(A-G) Live follow-up at 40 hpf of AGM cells labelled by laser-mediated uncaging of caged fluorescein-dextran at 26 hpf (A-D), or 38 hpf (E-G).
(A-D) Same embryo as in Figure 5C; labelled cells in the CHT have become numerous (A), their morphologies have become more diverse and they are found mainly in the abluminal mesenchyme (B-D).
(E-G) After labelling AGM cells at 38 hpf, labelled cells settle in the CHT within the next two hours, initially in the CV lumen.
(H-I) Following uncaging of rhodamine-dextran along the DP joint of CD41-gfp embryos at 52 hpf, the first red-labelled immigrants appear in the CHT two hours later, and are also GFP+, with early progenitor morphology.
Scale bars : 50 µm (A), 20 µm (E-G), 10 µm (B-D,H,I)
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