Mesp1 induces a subset of hematopoietic-associated transcription factors in ES cell–derived hemogenic endothelium. (A) ES cells derived from control heterozygous Mesp1^cre/+ mice (Mesp1^cre/+) or homozygous Mesp1-deficient Mesp1^cre/cre mice (Mesp1^cre/cre), as described previously,²⁴  were differentiated as EBs for 6 days and analyzed by FACS. Shown are 2-parameter histograms for expression of CD41 and Tie2. Numbers indicate the percentage of cells in the indicated quadrant. (B) ES cells described in panel A were cultured as EBs for 6 days before plating in methylcellulose media with cytokines as described in “Methods.” Hematopoietic colonies were quantitated after 6 days of growth on methylcellulose based on morphologies. Data represent the average of 3 experiments. Error bars represent SD. (C) ES cells harboring a doxycyline (dox)–inducible Mesp1 gene (A2lox.Mesp1) were differentiated as EBs for 5 days in the absence (−) or presence (+) of dox from day 2 to day 4. Flk1⁺ Tie2⁺ cells composing between 5% and 10% of the population (Presort) were purified by cell sorting (Postsort). (D) Microarray analysis of transcription factors associated with hematopoietic development. Expression of the indicated genes is shown as a ratio of expression values by dox-treated endothelial cells relative to untreated cells. (E) Cells described in panel A were cultured as EBs for 5 days, and total RNA was isolated to detect the expression levels of the indicated genes by quantitative RT-PCR using primers described in supplemental Table 1. (F) The 293T cells were transfected with firefly luciferase reporter constructs containing a minimal CMV promoter (CMVmini), CMVmini with Epha4 enhancer (Epha4), or 1 kb upstream promoter/enhancer regions for Meis1 (Meis1) and Meis2 (Meis2). These were cotransfected along with expression vectors for Mesp1 and E47, either separately or together as indicated. Luciferase activity was normalized using cotransfected Renilla luciferase construct (prL-CMV). Shown is the normalized luciferase for the indicated constructs. Bars represent the SD of triplicate determinations.