Treatment of Rps19 zebrafish morphants with L-leucine results in improved hemoglobinization. (A) Schematic of the experimental design. Embryos were injected at the single-cell stage with a morpholino targeting the translation initiation site of rps19, at 24 hours postfertilization (hpf) embryos were divided in half into 6-well plates and treated with 100mM L-leucine or egg water. (B-M) Representative images of zebrafish embryos at 4 days postfertilization (dpf) stained for hemoglobin with O-dianisidine. Lateral views with head to the left, dorsal upwards (B-E), ventral (F-I), and tail (J-M). Control morphants treated with egg water alone (B,F,J) showed normal hemoglobinization, as did the control morphants treated with 100mM L-leucine (C,G,K). rps19 morphants treated with egg water (D,H,L) were severely anemic and exhibited morphologic abnormalities including short body length, lack of bronchial arch development, and ventral tail kinking, all of which were alleviated with L-leucine treatment (E,I,M). (N) Quantification of the percentage of embryos exhibiting the phenotypes shown in panels B through M. There is a significant effect of L-leucine in the presence of Rps19 knockdown. The percentage of severely and moderately anemic rps19 morphants with leucine treatment are reduced, whereas those with normal hemoglobin or mild anemia are increased (***P < .001, 2-way ANOVA; error bars show SEM).