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Blood, Vol. 112, Issue 6, 2287-2296, September 15, 2008
spib is required for primitive myeloid development in Xenopus
Blood Costa et al.
112: 2287
Supplementary materials for: Costa et al
Files in this Data Supplement:
- Figure S1. Overlapping patterns of expression of spib and mpo in the Xenopus laevis stage 18 aVBI (JPG, 67 KB)
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Embryo was hybridized against digioxigenin-labelled spib anti-sense riboprobe and fluorescein-labelled mpo anti-sense riboprobe. The embryo was first developed with BCIP to reveal spib expression (light stain in embryo on left). Alkaline phosphatase activity was killed with 1hr incubation at 65°C in the presence of 0.1M EDTA, before subsequent detection of fluorescein-labelled mpo riboprobe with magenta phosphate (embryo on right). Overlap of domains of expression was revealed by a darker purple color, compared to magenta phosphate alone (inset, right top corner) or BCIP alone (left embryo). Same embryo is shown in left and right, before and after staining for mpo, respectively.
- Figure S2. Spib depletion (JPG, 62.5 KB)
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(A) Target sequences of the morpholino oligonucleotides used for spib depletion. (B) Analysis of sustained loss of neutrophils in stage 42 spib depleted tadpoles using sudan black staining.
- Figure S3. Loss of spib function does not alter the development and differentiation of the posterior ventral blood island (pVBI) (JPG, 64.2 KB)
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Erythroid differentiation is abundant in the pVBI of spib morphants. globin (A–C), scl (D–F) and lmo2 (G–M) WMISH on control and spib morphant embryos, markers of erythroid differentiation at stage 26. There is no shift to erythroid differentiation in the aVBI. The boundary between aVBI (white-myeloid) and pVBI (red-erythroid), and the characteristic pVBI V shape is maintained in spib morphant embryos (arrowheads). Anterior always to the left. (A–I) Ventral and (J–M) lateral views.
- Video 1. Ventral migration 20× (MOV, 3.69 MB)
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Time-lapse fluorescent microscopy movie illustrating the migration of primitive myeloid cells at stage 23 (end). Some cells show blebbing activity while others gained high motility and appear to follow pre-determined routes. Total imaging time of 208 minutes at a 20× magnification (2min/frame, 10frame/sec). Scale bar 10µm.
- Video 2. Lateral migration 4× (MOV, 4.82 MB)
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Lateral view of primitive myeloid cell migration at stage 26 (end). Elevated cellular motility is accompanied by cell shape elongation and extensive protrusions. Total imaging time of 6h20min at 4× magnification, (2 min/frame, 10frame/sec).
- Video 3. Onset migration (MOV, 4.56 MB)
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Time-lapse fluorescent video microscopy illustrating primitive myeloid cell migration away from a transplanted anterior ventral blood island (marked), in ventral view of a stage 20/1 embryo. At this stage “bleebing” is the most common cell behaviour. Total imaging time of 73 minutes at 30× magnification (2 min/frame, 10frame/sec).
- Video 4. Recruitment (MOV, 3.47 MB)
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Embryos previously transplanted with micro-ruby labelled anterior ventral blood islands (aVBI) as described above were infected with E. coli expressing GFP. Cells labelled in red are primitive myeloid progeny and the green area is injury and infection site made in the flank of the embryo. Inset from a time-lapse movie illustrating the recruitment of primitive myeloid cells to wound and infection sites well before the establishment of a functional circulatory system (stage 28). A white circle marks the location of wound and infected area. Total imaging time is 5h 35m at a 4× magnification, and a compression rate of 20 min per second.
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