Mesp1 rapidly promotes and is required for MCP specification and cardiac differentiation. (A) Schematic representation of Dox-inducible Mesp1 ESCs. (B) FACS analysis of the expression of CXCR4, PDGFRa, and Flk1 in Mesp1 Dox-inducible ESCs at D3, 24 h after Dox addition. (C) FACS quantification of CXCR4/PDGFRa/Flk1 TP cells in Mesp1 Dox-inducible ESCs 24 (D3) and 48 h (D4) after Dox addition. n = 3. (D and E) FACS quantification of proliferation (BrdU; D) and apoptosis (active caspase-3; E) in PDGFRa⁺/Flk1⁺ cells and in all Mesp1-inducible ESCs in the presence and absence of Dox for 24 h (D3). n = 2. (F) Schematic representation of Dox-inducible Engrailed (Engr)-Mesp1 ESCs (EN-Mesp1). (G) FACS analysis of CXCR4, PDGFRa, and Flk1 expression in EN-Mesp1–inducible ESCs at D4, 48 h after Dox addition. (B and G) Percentages of cells in each quadrant are shown, and the percentage of CXCR4/PDGFRa/Flk1 TP cells are shown in parentheses. (H) FACS quantification of TP cells in EN-Mesp1–inducible ESCs 24 (D3) and 48 h (D4) after Dox addition. Results are normalized to unstimulated cells. n = 3. (I) Quantification of beating areas in EN-Mesp1 ESCs in the presence or in the absence of Dox at D8. n = 3. (J and K) FACS quantification of cTNT (J) and CD31 (K) in EN-Mesp1–expressing cells. n = 3. Error bars indicate means ± SEM. TRE, tetracycline-responsive element. EB, embryoid body.