Figure 2
Figure 2. meunier (mnr) has miR-144/451 deficiency and an erythroid maturation defect despite normal erythroid specification. (A) Whole-mount in situ hybridization (WISH) expression analysis of miR-144, miR-451, miR-206, and gata1 at 24 hpf. mnr has normal gata1 expression (compared with wild-type Figure 1E) but is selectively deficient in the 2 erythroid miRNAs. Top panel mnr embryos were PCR genotype-confirmed; bottom panel is representative of 57 of 57 (gata1) and 32 of 34 (miR-206) embryos resulting from a mnr heterozygote incross. (B) Double WISH demonstrating that mnr has loss of miR-144 (loss of blue) despite retaining hbbe3 (red) expression; for comparison with WT, see Figure 1D. Left panel, bright field; right panel, fluorescence microscopy highlighting hbbe3 expression. (C) Normal expression of hemoglobin (brown) in mnr demonstrated by O-dianisidine (O-das) staining. At 48 hpf, mnr is recognizable by its smaller eye and head. Representative embryos are shown in lateral (left) and ventral (right) views to demonstrate equivalent O-das staining despite variation in the pattern of blood pooling at fixation. Embryos are representative of at least 30 embryos/genotype (see also Figure S3A). (D) Deficiency of mpx-expressing cells in mnr, displayed by a reduced number of EGFP-expressing cells in mnr embryos carrying the Tg(mpx:EGFP) reporter transgene (bottom panel) compared with wild type (top panel). Figure S4 shows expression of other hematopoietic and myeloid cell markers in mnr determined by WISH (tal1, spi1, lcp1, lyz, mpll). (E) Series of representative circulating erythrocytes from progressively older wild-type (WT + /?, top) and mnr (bottom) embryos, demonstrating the progressive maturation of WT erythrocytes and the persistent immature morphology of mnr erythrocytes. Erythrocytes are representative of the mean of groups tablulated in panel F. May-Grünwald/Giemsa stain; scale bars = 5 μm. (F) Tabulation of the N:C area ratio in WT and mnr embryos from 30 hpf to 4 dpf. The N:C ratio declines in both genotypes but is always greater in mnr than in WT. Data are mean plus or minus SD for n embryos, collected over 1 to 4 independent experiments. *P < .001 for WT compared with mnr. Figure S6 further demonstrates the reproducibility of these data by presenting them as scatterplots and displaying the interassay variation for the 48-hpf timepoint.

meunier (mnr) has miR-144/451 deficiency and an erythroid maturation defect despite normal erythroid specification. (A) Whole-mount in situ hybridization (WISH) expression analysis of miR-144, miR-451, miR-206, and gata1 at 24 hpf. mnr has normal gata1 expression (compared with wild-type Figure 1E) but is selectively deficient in the 2 erythroid miRNAs. Top panel mnr embryos were PCR genotype-confirmed; bottom panel is representative of 57 of 57 (gata1) and 32 of 34 (miR-206) embryos resulting from a mnr heterozygote incross. (B) Double WISH demonstrating that mnr has loss of miR-144 (loss of blue) despite retaining hbbe3 (red) expression; for comparison with WT, see Figure 1D. Left panel, bright field; right panel, fluorescence microscopy highlighting hbbe3 expression. (C) Normal expression of hemoglobin (brown) in mnr demonstrated by O-dianisidine (O-das) staining. At 48 hpf, mnr is recognizable by its smaller eye and head. Representative embryos are shown in lateral (left) and ventral (right) views to demonstrate equivalent O-das staining despite variation in the pattern of blood pooling at fixation. Embryos are representative of at least 30 embryos/genotype (see also Figure S3A). (D) Deficiency of mpx-expressing cells in mnr, displayed by a reduced number of EGFP-expressing cells in mnr embryos carrying the Tg(mpx:EGFP) reporter transgene (bottom panel) compared with wild type (top panel). Figure S4 shows expression of other hematopoietic and myeloid cell markers in mnr determined by WISH (tal1, spi1, lcp1, lyz, mpll). (E) Series of representative circulating erythrocytes from progressively older wild-type (WT + /?, top) and mnr (bottom) embryos, demonstrating the progressive maturation of WT erythrocytes and the persistent immature morphology of mnr erythrocytes. Erythrocytes are representative of the mean of groups tablulated in panel F. May-Grünwald/Giemsa stain; scale bars = 5 μm. (F) Tabulation of the N:C area ratio in WT and mnr embryos from 30 hpf to 4 dpf. The N:C ratio declines in both genotypes but is always greater in mnr than in WT. Data are mean plus or minus SD for n embryos, collected over 1 to 4 independent experiments. *P < .001 for WT compared with mnr. Figure S6 further demonstrates the reproducibility of these data by presenting them as scatterplots and displaying the interassay variation for the 48-hpf timepoint.

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