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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.
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