ER71 is inhibitory for cardiogenic development. (A) T/Brachyury (T/Bry) expression in sorted Flk-1⁺ mesoderm from day 3.5 Er71^+/+ or Er71^−/− EBs. (B) Flk-1⁺ mesoderm was sorted as in panel A and plated on OP9 cells. Cells were analyzed for CD41, CD45, CD31, and VE-cadherin (day 4) or cTnT and α-SMA expression (day 6). Representative results from 5 independent experiments are shown. (C) Flk-1⁺ mesoderm was sorted as in panel A, cultured on OP9, and analyzed for gene expression after 6 days. Expression levels relative to Gapdh are shown as means ± SD (n = 3). (D) Intracellular cTnT and SMAα staining of day 8 Er71^+/+ and Er71^−/− EBs are shown. (E) The frequency of Er71^+/+ and Er71^−/− EBs exhibiting spontaneous beating. Data shown are means ± SD (n = 3). **P < .05 compared with Er71^+/+ EBs. (F-J) Increased electrophysiological activities in spontaneously beating clusters from Er71^−/− EBs. Whole-cell current clamp recordings were obtained from spontaneously beating clusters in day 8 Er71^+/+ and Er71^−/− EBs. Vm in Er71^+/+ (F) and Er71^−/− (H) EBs, and maximal rates of rise of the spontaneous membrane depolarizations (dVmax/dt) and membrane depolarization amplitudes (ΔVmax) in Er71^+/+ (G) and Er71^−/− (I) EBs were measured. Mean values ± SEM are summarized in panel J. (K) iEr71 ES cells were differentiated in the presence (+DOX) or absence (−DOX) of DOX. Flk-1⁺ mesoderm was sorted and cultured on OP9. Cells were analyzed for CD31, VE-cadherin, cTnT, and SMAα expression, as described in panel B. (I) Flk-1⁺PDGFRα⁺ cells were sorted from day 3 iEr71 EBs and re-aggregated in the presence or absence of DOX. Flk-1 and PDGFRα expression were analyzed after 24 hours.