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Fig. 3. Activation of CE in ß-catenin-treated cells and node/notochord enhancer activity require two separate sequences within the CE. (A) Sequences of wild-type (WT) and mutated CEs (M1-5 and ins4/5). In the mutated CEs, the altered or inserted nucleotides are shown in red lower case. The nucleotides required for the activation are summarized in wild type sequence and denoted with red. (B) Expression of reporters containing wild-type and mutated 8xCEs 48 hours after co-transfection of the stabilized ß-catenin expression plasmid. (C) Transgenic embryos containing wild-type enhancer express ß-galactosidase in the node and notochord, reflecting endogenous Foxa2 expression accompanied by ectopic expression in the primitive streak region (n=9/11). (D,F) Transgenic embryos with mutated enhancers lacking activity in ß-catenin-expressing P19 cells did not show ß-galactosidase expression in the node or notochord (D: M1-2; n=0/8, F: M4; n=0/10). (E,G) Transgenic embryos with mutated enhancers with activity in P19 cells retained ß-galactosidase expression in the node and notochord (E: M3; n=3/7, G: M5; n=6/8). n, node; nc, notochord; ps, primitive streak.

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