Smad1 expression levels correlate with hemangioblast specification during ES/EB differentiation. (A) mSmad1 RNA expression in cells or colonies derived from the ES cell/EB system, or embryonic tissues. Ribosomal L32 expression levels serve as an internal control. Samples are as follows: Bra⁻Flk⁻, nonmesodermal early EB cells; Bra⁺Flk⁻, EB cells sorted for Brachyury (mesoderm population); Bra⁺Flk⁺, ES cells sorted for Brachyury and Flk1 (enriched for the hemangioblast population); LiqTR(− and +), transitional colonies from EB cells grown in the presence or absence of cytokines; Blast, blast (hemangioblast-derived) colonies; EryP, primitive erythroblast colonies; d14FL/EPO/mix, cells from day 14 fetal liver cultured without cytokines or with EPO (2 U/mL) only or with a mix of cytokines (EPO, 2 U/mL; kit ligand and IL-3, each 1% conditioned media; IL-11, 5 ng/mL; and TPO, 5 ng/mL); Mac, macrophage colonies; Endo, endothelial cells formed from day 6 embryoid bodies cultured with bFGF (100 ng/mL); D4T, an endothelial cell line; day 7, bottom and top, respectively, of whole day 7 mouse embryos; d8.5 (e and YS), day 8.5 mouse embryo and yolk sac, respectively; d9.5 (e and YS), day 9.5 mouse embryo and yolk sac, respectively. (B) Quantification of mSmad1 RNA expression in a subset of the samples from panel A normalized to the L32 control and graphed as fold change compared to Bra⁻Flk⁻. These are data from one representative experiment, but essentially the same results were obtained in at least 3 independent experiments. (C) The same as panel B, except analyzing relative Smad5 mRNA levels. Again, essentially the same results were obtained in at least 3 independent experiments.