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Blood, Vol. 113, Issue 8, 1794-1804, February 19, 2009
miR-451 regulates zebrafish erythroid maturation in vivo via its target gata2
Blood Pase et al.
113: 1794
Supplemental materials for: Pase et al
Files in this Data Supplement:
- Figure S7. Acridine orange staining for apoptotic cells in mnr embryos (JPG, 121 KB)
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mnr and sibling wild-type (WT) embryos at 24 and 48 hpf stained for apoptotic cells by acridine orange. At both ages, despite an excess of acridine-orange staining cells in the nervous system, there is no excess staining of hematopoietic cells. Embryos are lateral views with insets at higher power. Insets show higher power views of boxed areas. mnr embryos were recognized at 24 hpf by abnormal acridine orange staining of the brain, occurring in a Mendelian ratio; at 48 hpf, they were recognised by their characteristc gross appearance. C=circulation, ICM=intermediate cell mass, n=notochord, N=neural tube, S=somite, Y=yolk.
- Figure S8. Demonstration of activity of miRNA duplexes and morpholino oligonucleotide antisense miRNA inhibitors (JPG, 324 KB)
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(A) Sequences of miRNA reagents used in reagent validation assays of this figure. For each respective miRNA, the corresponding perfect target (PT) and antisense morpholino oligonucleotide (MO) sequences are shown. (B) Schema of a reporter assay to demonstrate the functional interaction of synthetic miRNA duplexes with 3UTRs containing their perfect target (PT) site in triplicate. One-cell embryos were injected with a sensor mRNAs encoding GFP with a 3UTR carrying triplicate perfect miRNA target sites, followed by separate injection of 50% of embryos with test miRNA duplex traced by rhodamine, and the impact on GFP fluorescence intensity assessed at 24–28 hpf. (C) Schema of a reporter assay to demonstrate the functional interaction of morpholino oligonucleotides (MO) designed to bind miRNAs and inhibit their interaction with 3UTR target sequences. One-cell embryos were injected with a bolus containing both the test MO and a sensor mRNA encoding GFP with triplicate perfect miRNA target sites in its 3UTR, followed by separate injection of 50% of embryos with the corresponding miRNA duplex traced by rhodamine, and the impact on GFP fluorescence intensity assessed at 24–28 hpf. (D, E) Bright field (upper panel), green fluorescence (middle panel) and red fluorescence (lower panel) images of embryo arrays assayed as in (B), demonstrating functional interaction of miR-144 (D) and miR-451 (E) and their perfect recognition site, resulting in reduced GFP fluorescence intensity. Note that wild-type embryos remained phenotypically normal despite the ectopic- and over-expression of each miRNA. (F, G) Bright field (upper panel), green fluorescence (middle panel) and red fluorescence (lower panel) images of embryo arrays assayed as in (C), demonstrating functional interaction of MO-144 (F) and MO-451 (G) with their respective miRNA duplex, resulting in blockage of the duplex-induced GFP fluorescence intensity reduction. Note that wild-type embryos were phenotypically normal after microinjection of MO-144 and MO-451. In (C–F), arrays of representative embryos were photographed together in single images to ensure valid comparison of relative fluorescence intensity between the two groups.
- Figure S9. Supplementary data corresponding to Fig. 3A, showing that miR-451 deficiency, but not miR-144 deficiency, causes erythrocyte immaturity (JPG, 174 KB)
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(A) Scatterplots of the N:C ratio in 2 days post-fertilization (dpf) wild-type (WT) embryos injected with a control morpholino oligonucleotide (MO-Con) or morpholino antagonists of miR-451 (MO-451) or miR-144 (MO-144). The 4 colors (blue, red, green, purple) indicate data from 4 independent assays. horizontal lines indicate mean values. These data correspond to the respective groups tabulated in Fig. 3A. (B) Representative fields showing erythrocytes from 2 dpf WT embryos injected with either MO-451 or MO-144. Although the effect of MO-451 on N:C ratio may be subtle, the erythrocytes also display other features of immaturity: larger diameter, a more open pattern of nuclear chromatin, and a more basophilic cytoplasm. May-Grünwald/Giemsa stain; scale bar=5 µm.
- Figure S10. Supplementary data corresponding to Fig. 3D–E, showing that overexpression of miR-451, but not miR-144, is sufficient to partially rescue the erythroid maturation block in mnr (JPG, 73.4 KB)
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(A) Scatterplots of the N:C ratio in 2 days post-fertilization (dpf) wild-type (WT) embryos or mnr embryos injected with control RNA duplex (miR-C), RNA duplex mimicking miR-144 (miR-144), RNA duplex mimicking miR-451 (miR-451) or both miR-144 and miR-451 duplexes (miR144+451). In the presence of miR-451, the mnr erythrocyte N:C ratio is reduced towards that of WT. The 4 colors (blue, red, green, purple) indicate data from 4 independent assays; horizontal lines indicate mean values. These data correspond to the respective groups tabulated in Fig. 3E. (B) Representative fields showing erythrocytes from 2 dpf WT embryos injected or mnr embryos injected with miR-C, miR-144, MO-451 or miR-144+451. Not only do mnr embryos injected with miR-451 have erythrocytes with a partially rescued N:C ratio towards WT (Fig. S10A above), but their erythrocytes (black boxed panel) display a morphology indicative of a intermediate state between mnr erythrocytes and miR-C or miR-144–injected mnr erythrocytes (blue boxed panel) and WT erythrocytes of any group (panels above). In particular, compared to mnr, some cells have a smaller diameter overall, and more densely compacted nuclear chromatin. Fig. 3D shows higher power views of single erythrocytes representative of cells in the fields shown here. May-Grünwald/Giemsa stain; scale bar=5 µm.
- Figure S11. Supplementary data corresponding to Fig. 5C, showing that gata2 knockdown in mnr is sufficient to partially restore erythrocyte maturation (JPG, 229 KB)
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(A) Scatterplots of the nuclear:cytoplasmic (N:C) area ratio in 2 days post-fertilization (dpf) mnr embryos injected with a gata2 morpholino antisense oligonucleotide (gata2 MO) or control MO. Knockdown of gata2 in mnr reduces the N:C ratio of erythrocytes toward wild-type to an extent similar to that achieved by overexpressing miR-451 in mnr (Fig. S10A). The 3 colors (blue, red, green) indicate data from 3 independent assays; horizontal lines indicate mean values. (B) Representative fields showing erythrocytes from 2 dpf mnr embryos injected or mnr embryos injected with gata2 MO or control MO. Not only do mnr embryos injected with gata2 MO have erythrocytes with a partially rescued N:C ratio towards WT (Fig. S11A above), but their erythrocytes display a morphology indicative of a intermediate state between mnr and either WT, miR-C or miR-144 injected mnr erythrocyte: generally cells have a smaller diameter overall, and more densely compacted nuclear chromatin. Fig. 5C shows higher power views of single erythrocytes representative of cells in the fields shown here. May-Grünwald/Giemsa stain; scale bar=5 µm.
- Figure S12. Supplementary data corresponding to Figure 6F, showing that gata2 target-blocking morpholino oligonucleotides (MO-TB) do not effect erythrocyte maturation (JPG, 126 KB)
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(A) Scatterplots of the nuclear:cytoplasmic (N:C) area ratio in 2 days post-fertilization (dpf) wild-type(WT) embryos injected with a gata2 TB-MO designed to interfere with the miR-451 interaction with its binding sites a and b in the 3′UTR of gata2 (MO-TB site a+b) or a control gata2 3′UTR binding MO (MO-TB control). MO-TB design is detailed in Fig. 6A. There was no significant difference in the erythrocyte N:C ratio between the MO-TB site a+b and MO-TB control groups. The 3 colors (blue, red, green) indicate data from 3 independent assays. horizontal lines indicate mean values. (B) Representative fields showing erythrocytes from 2 dpf WT embryos injected with MO-TB site a+b or MO-TB control. Not only is the N:C ratio the same, but the eyrthocytes have similar morphology. May-Grünwald/Giemsa stain; scale bar=5 µm.
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