Effect of morpholino knockdown of transferrin-a or TfRs on gene expression. Transferrin-a expression was reduced after morpholino knockdown of transferrin-a (A). Hepcidin expression was also reduced after knockdown of transferrin-a (B), but not after injection of a mismatch morpholino with 5 nucleotide substitutions (C). Knockdown of TfR2 (D) resulted in low hepcidin expression that failed to increase significantly after iron dextran injection. In contrast, knockdown of TfR1a (E) or TFR1b (F) did not impair baseline hepcidin expression, although the response to iron injection was blunted in TfR1a morphants. (G-J) Whole mount in situ hybridization at 72 hpf, in the absence of iron injection. Decreased hepcidin expression was observed in TfR2 morphant embryos (G), while knockdown of TfR1a and TfR1b in combination (H) failed to reduce hepcidin expression. Producing anemia by treatment of embryos with phenylhydrazine (I) or morpholino knockdown of GATA1 (J) resulted in strong hepcidin expression. White arrow marks the liver. *P < .05 compared with embryos that were neither injected with iron dextran nor morpholino.