Fig. 1. Characterization of methylcellulose colonies. (A) Micrographs of representative colonies from methylcellulose cultures: 2° EB colony (A '), transitional colony (Trans-CFC; D') and hemangioblast colony (BL-CFC; G'). Microwells of colonies replated on matrigel: 2° EB with adherent mesodermal cell type (B',C'); two independent transitional colonies showing mostly hematopoietic cells (E',F'); BL-CFCs showing both adherent endothelial cells (arrows) and overlaying non-adherent hematopoietic cells (arrowhead; H',I'). (B) Single hemangioblasts were replated on matrigel for one week and treated with fluorescent DiI-Ac-LDL. Phase contrast (left panel) and fluorescent micrograph (right panel) images are shown. (C) RNA from individual Arnt^+/+ and Arnt^–/– 2° EB, Trans-CFC and BL-CFC colonies were analyzed for the expression of the indicated genes by radioactive RT-PCR: ß-actin (control), Rex1 (ES cells), Bry (mesoderm), Scl (endothelial/hematopoietic cells) and GATA1 (hematopoietic cells). As BL-CFCs are generally smaller colonies, the signal for ß-actin is lower.