Arhgef3 is essential for late stages of erythroid differentiation. (A-B) Whole-mount in situ hybridization showing arhgef3 expression in Danio rerio embryos at 24 hpf. (A) Arhgef3 was expressed in eyes, brain, and ICM of the developing embryos. (B) Higher magnification view of the boxed area in (A) shows the expression of arhgef3 in the ICM (nc indicates notochord; pd, pronephric duct). (C) Quantitative real-time PCR analysis of arhgef3 expression in gata1⁺ and fli1⁺ cells sorted by FACS. Arhgef3 mRNA expression was normalized to β-actin expression. Results are presented as quotient of Ct values for arhgef3 and β-actin ± SD. (D-E) O-Dianisidine staining of hemoglobin was used to assess the number of mature erythrocytes in arhgef3 MO-injected embryos. At 30 hpf, hemoglobin-positive cells were present on the yolk and in circulation of control embryos (D and D″ black arrows); however, arhgef3 MO-injected embryos completely lacked such cells at the same time point (E,E″). At 48 (G,G′) and 72 (I,I′) hpf, the number of o-Dianisidine–positive cells in arhgef3 MO-injected embryos was severely reduced compared with time-matched controls (F,F′ and H,H′, respectively). (D′-E″) TUNEL assay in control- and arhgef3 MO-injected embryos at 24 hpf. There was no change in the number of apoptotic cells in the ICM in the arhgef3 MO-injected (E′) compared with control embryos (D′). Insets in (D′) and (E′) show 1 TUNEL-positive cell under higher magnification.