Fig. 1. Gene expression analysis of developing blast colonies. Flk1⁺ cells isolated from Bry-GFP ES cell-derived day 3.25 EBs were cultured in serum-containing hemangioblast methylcellulose media and the developing blast colonies assayed at the indicated time intervals. (A) Images showing the morphology of representative developing blast colonies over a 72-hour culture period. Scale bars: 10 µm. (B) Expression profiles of individual developing blast colonies isolated at the indicated times. Each lane represents an individual colony. Pos, positive control. (C) Hematopoietic progenitor potential of different aged blast colonies. Seven-hundred colonies of each time point were collected, dissociated and the cells plated in methylcellulose media supplemented with hematopoietic cytokines. Primitive erythroid (Ep), macrophage (Mac) and bipotential macrophage/erythroid (Emac) colonies were scored after 4-5 days of culture, while definitive erythroid (Ed) and multipotential myeloid/erythroid (Mix) colonies were scored at day 9 of culture. (D) Expression of Notch and Wnt signaling components in developing blast colonies revealed by quantitative PCR. 3' cDNA samples of day 3.25 Flk1⁺ cells (0 hours) and 3' cDNAs of seven individual blast colonies used in B were pooled and analyzed at each time point (12-48 hours). Average expression normalized to Actb is shown. Error bars represent the standard errors of mean from three independent experiments.