gata2 and miR-451 interact in vivo to regulate erythrocyte maturation. (A) gata2 expression (blue) in 24- and 27-hpf embryos by WISH arranged for side-by-side comparison. Left panels (i, iii, v): in wild-type (WT), MO-144- and MO-control-injected embryos, gata2 expression is waning in the anterior intermediate cell mass at 24 hpf (▿). Right panels (ii, iv); in miR-451-deficient mnr and MO-451-injected morphants, gata2 expression in the anterior intermediate cell mass persists at 24 hpf (▾). (vi,vii) To examine the duration of the persistence of gata2 expression, a time course of gata2 expression was examined by WISH at 27-, 30-, 36- and 48-hpf timepoints. At the onset of circulation (27 hpf), sustained gata2 expression (> WT) was still evident in mnr erythocytes in the anterior intermediate cell mass and over the yolk (vii, solid arrowheads). i and ii are unmanipulated age-matched siblings and representative of 19 of 19 and 11 of 12 PCR-genotype confirmed WT and mnr embryos, respectively. Subpanel i, ii, vi, and vii embryos are PCR-genotype confirmed. Subpanels iii through vii are representative of more than 40 age-matched embryos. See Figure 6E legend for further details about controls for comparing the level of gata2 expression. (B) Schematic diagram of an experiment testing if gata2 knockdown by a gata2 MO affects erythrocyte maturation. (C) Results of the experiment in panel B, showing that gata2 knockdown in mnr is sufficient to partially restore erythrocyte maturation (both morphologically and as measured by N:C area ratio). Data are mean plus or minus SE for n independent experiments. *P = .04, 2-tailed t test. Scale bar = 5 μm. Figure S11 further demonstrates the reproducibility of these data by presenting them as scatterplots and providing additional fields of representative cells.