Sox7 is expressed at the transition from hemangioblast to CD41 up-regulation. (A) Sox7-GFP expression in day 3 EBs derived from an ES cell line transgenic for the Sox7-GFP BAC Construct. WT indicates wild type. (B) Expression of endogenous Sox7 was assessed by RT-PCR on GFP⁺ and GFP⁻ cells sorted from day 3 wt and Sox7-GFP EBs. (C) Flow cytometric analysis of day 3 Sox7-GFP EBs for Flk1 and CD41 expression. (D) Cells from day 3 Sox7-GFP EBs were sorted based on Flk1 and GFP expression and replated in clonogenic assay for hemangioblast progenitors. (E) Sorted Flk1⁺Sox7-GFP⁻ cells were cultured for 24 hours in liquid hemangioblast mix then analyzed for Sox7-GFP⁺ expression. Flk1 staining after 24 hours represents remaining staining from sort. (F) Sorted Flk1⁺Sox7–GFP⁺ cells were cultured for 24 or 48 hours in liquid hemangioblast mix then analyzed for CD41 and Sox7–GFP expression. (G) After 48 hours of culture, wt Flk1⁺ cells, Flk1⁺Sox7-GFP⁻, and Flk1⁺Sox7-GFP⁺ cells were tested for the presence of hematopoietic progenitors in semisolid clonogenic replating assay. All data are representative of at least 3 independent experiments. Error bars, where visible, represent SEM.