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Blood, Vol. 114, Issue 1, 40-48, July 2, 2009
C/EBP initiates primitive myelopoiesis in pluripotent embryonic cells
Blood Chen et al.
114: 40
Supplemental materials for: Chen et al
Files in this Data Supplement:
- Table S1. RT-PCR primer and Q-PCR probe sequences (PDF, 18.7 KB)
- Figure S1. Spatial expression pattern of cebpa and gsc at stage 12 (JPG, 140 KB)
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Whole mount in situ hybridization analysis of cebpa (A–C) and gsc (D–F), showing different views of the same embryos. Panel A and D are anterior views and Panel B and E are dorsal views. Panel C and F are images of the same embryos shown in A and C, respectively, which have been transected in half. The images show that the both genes are expressed within the dorsal/anterior mesoderm. Panel C and F have dorsal left, anterior up and blastopore (posterior) at the bottom.
- Figure S2. Western blots of embryo lysates expressing various C/EBPα constructs in the presence or absence of the cepbaATG MO (JPG, 89.6 KB)
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Co-injecting 10ng cebpaATG MO inhibits expression of wild type C/EBPα (lane 4) but not HA-C/EBPα (lane 6). Antibodies against C/EBPα or HA were used as probes. Endogenous C/EBPα is not detected under these conditions.
- Figure S3. Loss of function of cepba does not alter the expression of scl, fli1, and globin (JPG, 281 KB)
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(A) Expression of scl, fli1, and globin in cebpaATG MO injected Xenopus tropicalis embryos, at stage 23, was not affected. All images are lateral views, anterior is to the left. (B) Whole embryo real-time PCR at stage 23 showed no significant change of fli1, scl, and globin. Expression levels were normalized to rpl8. Error bars represent standard error mean (S.E.M) of four independent experiments. Statistical ANOVA analysis was done using the SPSS software package. The statistic tests showed that none of the differences was significant.
- Figure S4. Ectopic expression of hematopoietic markers induced by C/EBPα (JPG, 289 KB)
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Embryos injected with 10pg cebpa mRNA into one cell at 8-cell stage were fixed at stage 18 and assayed by whole mount in situ hybridization against spib (A, B); mpo (C, D); lcp (E, F); mmp7 (G, H); spi1 (I, J); fli1 (K, L) and scl (M, N). The expression of all examined primitive myeloid markers and hemangioblast markers were detected in areas outside their normal expression domains. A, B, G–N: lateral view, anterior to the left, dorsal to the top; C, D: ventral view, anterior to the left; E, F: dorsal view, anterior to the left.
- Video 1. Time-lapse fluorescence video microscopy of embryos with transplanted patches of animal cap tissue expressing eGFP (L) or cebpa/eGFP (R) during the gastrula to neurula stages (MOV, 4.5 MB)
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The GFP positive control transplant on the left went through normal epiboly and kept a clear boundary with the host cells of the embryo. In contrast, the GFP/C/EBPα transplant show many migratory GFP positive cells emerging from the transplant. Total imaging time of 11 hour 40 minutes (2 min/frame, 36 frame/second) at a magnification of 63×.
- Video 2. Time-lapse fluorescence video microscopy of cells expressing cebpa/eGFP at stage 40 (tadpole stage) (MOV, 3.57 MB)
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The cells, which were derived from transplanted animal cap cells, show highly active migration. Total imaging time of 6 hours 30 minutes (1.5 min/frame, 15 frame/second) at a magnification of 100×.
- Video 3. Time-lapse fluorescence video microscopy of cells expressing cebpa/eGFP, showing that a subset of GFP positive cells enter the circulation (MOV, 2.33 MB)
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The arrow points at a blood vessel. Total imaging time of 1 hour 15 minutes (45 sec/frame, 15 frame/second) at a magnification of 63×.
- Video 4. Time-lapse fluorescence video microscopy of cells expressing cebpa/eGFP, showing random migration before wounding (MOV, 2.21 MB)
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Lateral view of a stage 34 chimera with C/EBPα misexpressing cells derived from animal cap transplantation. Note that only a subset of cebpa/eGFP is migratory, while other cebpa/eGFP expressing cells retain the morphology of epidermal cells. This is the same embryo shown in Video 5. Total imaging time of 1 hour 50 minutes (1.5 min/frame, 6 frame/second) at a magnification of 63×.
- Video 5. Time-lapse fluorescence video microscopy of cells expressing cebpa/eGFP, showing directed migration toward wound (MOV, 6.31 MB)
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Embryo shown in Video 4 was wounded and imaged immediately after. Wound is marked by a circle in the first image. The video shows that many graft derived GFP positive cells immediately respond to the wound by migrating towards it. Total imaging time of 8 hours (1.5 min/frame, 12 frame/second) at a magnification of 63×.
- Video 6. Time-lapse fluorescence video microscopy of control cells expressing GFP after wounding (MOV, 2.99 MB)
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An embryo containing control GFP positive animal cap cells gave rise to epidermis and showed stretching behaviour towards the wound site but no active migration. Wound is marked by circle in the first image. Total imaging time of 6 hours (1.5 min/frame, 26 frame/second) at a magnification of 63×.
- Video 7. Time-lapse fluorescence video microscopy of cells expressing cebpa/eGFP, showing phagocytosis of mCherry-labeled bacteria (MOV, 6.52 MB)
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This video was taken on a standard fluorescent microscope, which does not allow the simultaneous visualization of different fluorescent channels. Thus the initial frames show the site of red bacterial infection. Then the video ensues in the green channel, where the cebpa/eGFP expressing cells become apparent. These cebpa/eGFP positive cells immediately migrate toward the site of infection, and as the video progresses (and the red channel flashes), one can see that the green cells take up the red bacteria and continue migrating around the infection site. Total imaging time of 22 hours (2 min/frame, 20 frame/second) at a magnification of 63×.
- Video 8. Time-lapse confocal fluorescence video microscopy of cells in the ventral fin of a stage 41 Xenopus laevis tadpole, 24 hours after bacterial infection (MOV, 4.6 MB)
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The video shows the behaviour of endogenous and cebpa induced myeloid cells, 24 hours after infection. Patrolling cebpa/eGFP positive cells, which contain yellow areas within them, are those that have phagocytosed the red bacteria. Endogenous myeloid cells that have engulfed bacteria are seen in red and cebpa/eGFP cells that have not engulfed bacteria are seen in green. Note that one of the green cells in this video rounds up and divides (middle upper). Also note the ramified morphology of the cells, reminiscent of differentiated macrophages. The video shows a maximum intensity projection of 10 z-sections over 50µm every 2 minutes over 2 hours and played at 20 fps, created using Imaris software (Bitplane, Zurich).
- Video 9. Three-dimensional time-lapse view of the changes in morphology and behavior of primitive myeloid cells over time (MOV, 4.36 MB)
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This video was generated from the same time-lapse confocal stacks shown in Video 8, but processed using Imaris software to obtain a higher resolution, 3-D time-lapse view of the cells’ morphology and behavior. The green semi-transparent 3D volumes represent signal from cebpa/eGFP positive cells, and the red channel represent the mCherry expressing bacteria. Note that the red bacteria are contained within vacuoles in the cebpa/eGFP positive cells and the movement of the vacuoles within the cells as they move. Also note that one of the cebpa/eGFP cells in the centre rounds up and divides. The 3D iso-volume reconstruction of a 50µm stack taken every 2 minutes over a period of 2 hours, played at 3fps was done using Imaris software (Bitpane, Zurich). The background grid spacing is of 50µm.
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