Fig. 4. Expression of E-cadherin and GFP in differentiating Mixl1 wild-type (Mixl1^w/w), heterozygote (Mixl1^GFP/w) and null (Mixl1^GFP/GFP) ES cells. (A) Flow cytometric analysis of day 3 EBs showed that more than 98% of cells were E-cadherin positive and more than a third of these also expressed GFP from the Mixl1 locus. (B) Time course of E-cadherin and GFP expression in differentiating Mixl1^GFP/W ES cells. Initially, E-cadherin-positive (E-cad⁺) stem cells differentiate into a transient double-positive population (E-cad⁺ GFP⁺) corresponding to cells at the primitive streak stage of development. After day 3, Mixl1-expressing (GFP⁺) nascent mesoderm downregulated E-cadherin.