Asymmetric template strand segregation is associated with asymmetric cell fate. (a) Paired-cell assays were performed on H9-derived EBs 7 d after differentiation using the BrdU single-labeling method. Cell pairs were immunostained for BrdU and the mesoderm marker Brachyury (Bry) or the endoderm marker Gata4. Representative photographs of paired cells symmetric (top) or asymmetric (bottom) for BrdU and expressing Bry or Gata4 are shown. (b and c) Data from experiments as in a were quantified. (b) Paired cells symmetric for BrdU were distinguished based on Bry (top) or Gata4 (bottom) expression pattern: symmetric and coincident with BrdU or asymmetric. (c) Paired cells asymmetric for BrdU were distinguished based on Bry (top) or Gata4 (bottom) expression pattern: symmetric, asymmetric, and coincident with BrdU or asymmetric and mutually exclusive with BrdU. More than 100 pairs were quantified for Bry and GATA4 for each experiment. Data represent means ± SEM (n = 3 independent experiments). BF, bright field. **, P < 0.01; ***, P < 0.001.