RPS19-deficient embryos have defects in erythropoiesis. (A) RT-PCR, red blood progenitors sorted out from embryos at 19 to 20 hpf. Globin expression is lower in morphants. The cells were purified from a pool of 300 embryos. (B) The differentiation of erythroid cells is delayed in morphants: at 30 hpf almost all erythroid cells are still in ICM region in morphants. In situ hybridization, globin αe1 probe, 30 hpf, 30 embryos per group. (C) Expression of c-myb in the tail regions in morphants () points to immaturity of red blood cells in these embryos. In situ hybridization, 30 hpf. (D) At 72 hpf, there are fewer blood cells in morphants; they are localized mostly in the posterior of the embryo (), and there are only few cells in the heart region: 2.5 ng morpholino per embryo, O-dianizidine staining, 30 embryos per group. (E) Erythroid cells from the blood of a morphant embryo are variable in size; some look like erythroblasts (). Zebrafish erythrocytes differ from mammalian cells in that both primitive and definitive cells are nucleated. Blood smears were prepared from individual embryos; the results are representative of 6 to 10 embryos. (F) The level of hemoglobin is lower in morphants at day 5 in dependence with morpholino dose. Hemoglobin levels were measured in pools of 80 embryos in triplicates. Five-day-old embryos (wild-type, morphants, or cloche mutants, which have no blood cells) were homogenized in 500 μL of Drabkin reagent (Sigma-Aldrich, St Louis, MO), incubated for 1 hour at room temperature, centrifuged 10 minutes at 16 000g, and the absorbance of supernatants was measured at 540 nm. Absorbance of cloche mutants was subtracted from the wild-type and morphant samples. In this way, only the absorbance derived from red blood cells was taken into consideration. It was plotted on a calibration curve obtained from a standard sample with known concentration of hemoglobin (Drew Scientific, Dallas, TX), and the amount of hemoglobin per embryo was calculated.