Fig. 6. Molecular program at the onset of hemangioblast specification. (A) ES cells were induced to differentiate in serum-free culture conditions with Bmp4 for 6 days (lanes B), Bmp4 followed by activin A and bFGF at day 2.5 (lanes BAF) or Bmp4 followed by activin A, bFGF and VEGF at day 2.5 (lanes BAFV). Day 0, 1 and 2: Bmp stimulation only for the three conditions. Equal amounts of RNA from ES cells or from EB-derived cells were reverse-transcribed and analyzed by RT-PCR for the expression of the indicated genes. (B,C) EBs grown for 2.5 days in serum-free culture with 4 ng/ml Bmp4 were further stimulated with either activin A and bFGF (AF) or with activin A, bFGF and VEGF (AFV) and harvested 1, 2, 3, 6 or 24 hours after stimulation for gene expression analysis by real-time PCR. cDNA derived from EBs grown in serum condition for 4.5 days were used as positive control (C+). All real-time PCR data are presented as mRNA expression level relative to that of β-actin. ^*A statistically significant difference (P<0.05) compared with Bmp4-stimulated control at 0 hour. Data are mean±s.e.m. of triplicate values from three independent experiments. (D) Schematic representation of the differentiation steps leading to the formation of hematopoietic progenitors. All data shown are representative of at least three experiments.