Fig. 2. ARNT is required for the appropriate development of hemangioblasts, and hypoxia accelerates the kinetics of their appearance. ES cell lines were differentiated into EBs and replated in a BL-CFC methylcellulose assay. (A) Five independent Arnt^–/– clones (patterned bars) were differentiated for 2.75 days at 21% O₂, replated and compared with the wild-type control (black bar). (B) Arnt^+/+ (left panels) and Arnt^–/– (right panels) ES lines were differentiated into EBs under normoxic (white bars, N; 21% O₂) or hypoxic (black bars, H; 3% O₂) conditions, for the indicated times, and replated for hemangioblast analysis. The number of transitional colonies is stimulated by hypoxia in Arnt^+/+ cultures (days 2 and 2.5), but mutants are not affected (upper panels). Hypoxia also accelerates the kinetics and increases the number of BL-CFCs for wild-type cells but not for Arnt^–/– clones (lower panels). Error bars represent the standard error of the mean (s.e.m.) for triplicate assays. (C) Kinetics of Arnt^+/+ BL-CFC formation from 2 to 3.5 days, comparing normoxic (blue) and hypoxic (red) conditions.