Figure 3
Figure 3. Functional studies of the role of miR-451 in erythroid maturation. (A) miR-451 deficiency, but not miR-144 deficiency, is sufficient to cause erythroid immaturity. MO miRNA antagonists or control MO was microinjected, tracing delivery by rhodamine (schematic diagram) and the N:C area ratio computed as an indicator of maturation (table). O-diansidine staining confirmed normal hemoglobinization in MO-injected embryos at 48 hpf (compare Figure 2C); embryos in panels are representative of at least 16 embryos/group (see Figure S3B). MO reagent validation experiments are in Figure S2. Only MO-451 increased the N:C ratio, reflecting delayed erythrocyte maturation, in a dose-dependent manner. Data are mean plus or minus SE for n independent groups from 3 separate experiments. *P = .036; †P = .048 for the comparisons of MO-451-injected with control-injected and MOmiR-144-injected groups, respectively, 2-tailed t test. Figure S9 further demonstrates the reproducibility of these data by presenting them as scatterplots and providing additional fields of representative cells. (B) Knockdown of miR-144/451 does not affect neutrophil numbers and does not replicate the deficiency of mpx-expressing cells in mnr. Experiments were performed using WT or mnr embryos on the Tg(mpx:EGFP) background. Concentration of each MO injected was 1 mmol/L. Neutrophils were quantified by counting EGFP-positive cells/embryo at 48 hpf. *P < .0001 for comparison of mnr with all 4 other groups. (C-E) Overexpression of miR-451, but not miR-144, is sufficient to partially rescue the erythroid maturation block in mnr, as evident morphologically (D) and quantitatively (E) using the N:C area ratio. (C) A schema of the experimental design, which required genotyping embryos by nonhematologic phenotypic features at 48 hpf. Data are mean plus or minus SE for n independent experiments; ‡P < .0025 for line-by-line comparisons of WT to mnr, and §P = .035 and §§P = .003 for the indicated comparisons of miRNA-injected mnr with miR-C-injected mnr, 2-tailed t test. Scale bars = 5 μm. Figure S10 further demonstrates the reproducibility of these data by presenting them as scatterplots and providing additional fields of representative cells.

Functional studies of the role of miR-451 in erythroid maturation. (A) miR-451 deficiency, but not miR-144 deficiency, is sufficient to cause erythroid immaturity. MO miRNA antagonists or control MO was microinjected, tracing delivery by rhodamine (schematic diagram) and the N:C area ratio computed as an indicator of maturation (table). O-diansidine staining confirmed normal hemoglobinization in MO-injected embryos at 48 hpf (compare Figure 2C); embryos in panels are representative of at least 16 embryos/group (see Figure S3B). MO reagent validation experiments are in Figure S2. Only MO-451 increased the N:C ratio, reflecting delayed erythrocyte maturation, in a dose-dependent manner. Data are mean plus or minus SE for n independent groups from 3 separate experiments. *P = .036; †P = .048 for the comparisons of MO-451-injected with control-injected and MOmiR-144-injected groups, respectively, 2-tailed t test. Figure S9 further demonstrates the reproducibility of these data by presenting them as scatterplots and providing additional fields of representative cells. (B) Knockdown of miR-144/451 does not affect neutrophil numbers and does not replicate the deficiency of mpx-expressing cells in mnr. Experiments were performed using WT or mnr embryos on the Tg(mpx:EGFP) background. Concentration of each MO injected was 1 mmol/L. Neutrophils were quantified by counting EGFP-positive cells/embryo at 48 hpf. *P < .0001 for comparison of mnr with all 4 other groups. (C-E) Overexpression of miR-451, but not miR-144, is sufficient to partially rescue the erythroid maturation block in mnr, as evident morphologically (D) and quantitatively (E) using the N:C area ratio. (C) A schema of the experimental design, which required genotyping embryos by nonhematologic phenotypic features at 48 hpf. Data are mean plus or minus SE for n independent experiments; ‡P < .0025 for line-by-line comparisons of WT to mnr, and §P = .035 and §§P = .003 for the indicated comparisons of miRNA-injected mnr with miR-C-injected mnr, 2-tailed t test. Scale bars = 5 μm. Figure S10 further demonstrates the reproducibility of these data by presenting them as scatterplots and providing additional fields of representative cells.

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