Arhgef3 regulates iron uptake through activation of rhoad and rhoae. (A-C) Knockdown of rhoad/rhoae (C), but not rhoaa/rhoac (B), results in severe anemia compared with control embryos (A) as shown by o-Dianisidine staining at 48 hpf. (D-F) O-Dianisidine staining of rhoad and rhoae MO-injected embryos at 48 hpf shows a reduction in the number of hemoglobin-positive cells, which is more profound in rhoae-depleted embryos. (G-J) Provision of intracellular iron rescues the anemia of rhoad/rhoae-depleted embryos. Note o-Dianisidine–positive cells (indicated with black arrow) in the posterior blood island of embryos coinjected with rhoad/rhoae MO and iron dextran (Fe; J). (G) Graph to illustrate the difference (on the y-axis) between the percentages of embryos with hemoglobin positive (Hb⁺) erythrocytes. At 30 hpf, Hb⁺ cells were detected in 96% ± 0.9% of the control embryos (n_embryos = 53; G,H), whereas only 39% ± 6.6% of rhoad/rhoae MO-injected ones (n_embryos = 52) had Hb⁺ cells (G,I). Coinjection of rhoad/rhoae MO with Fe resulted in a significant increase in the number of embryos showing Hb staining (64% ± 3.2%; n_embryos = 64; G,J) compared with the rhoad/rhoae-depleted embryos (*P < .05 in graph). Error bars represent the SEM. (J) Inset shows higher magnification view of the boxed area; black arrow shows accumulated erythrocytes stained with o-Dianisidine.