Smad1 knockdown enhances erythropoiesis. (A-C) Fertilized eggs derived from the gata1:DsRed reporter line were either uninjected controls (A) or injected with S1MO (B) or S5MO (C). There is an increase in erythrocyte production in the tail of smad1 morphants at 48 hpf (B), while smad5 morphants have a significant decrease in DsRed⁺ cells (C). Representative embryos are shown, and approximately 200 of each type of embryo were observed. (D) These phenotypes are quantified by flow cytometry of cells derived from control or morphant embryos at 32 hpf. Shown is the percentage of DsRed⁺ cells compared with control wt. *The difference compared with wt is significant by Student t test; P < .001. For each of 4 experiments, 30 embryos of each phenotype were collected for flow cytometry. (E-G) In situ hybridization was carried out for βe3 globin on wild-type (E), smad1 morphants (F), and pgy/pgy embryos (G). Shown are representative embryos at 32 hpf, showing that smad1 morphants are increased in erythrocyte production at 32 hpf (F); in contrast, piggytail (Smad5) mutants have a decrease in βe3 globin expression (G). In situ hybridization was done on a sample of 25 embryos for each; panels E-G are representative of the phenotypes seen. (H) Quantitative real-time PCR for βe3 globin was carried out on smad1 (S1MO) and smad5 (S5MO) morphant embryos at 32 hpf. Shown is the average fold change in expression calculated from 4 independent experiments, with samples analyzed each time in triplicate. Samples were normalized to β-actin, and wt set to 1. *The difference compared with wt is significant by Student t test; P < .001. Error bars in D and H indicate the standard deviation from the mean.