(A) A 300 bp promoter region (schematized at top) containing the putative Lef-1 binding site CTTTGAA is sufficient to drive expression of a β-galactosidase reporter in the E lineage. Anti-β-galactosidase antibodies stain the daughters (2E) and granddaughters (4E) of the E blastomere in transgenic embryos expressing the reporter driven by this sequence.
(B) Electrophoretic mobility shift assays performed with extracts from developing early embryos detect a band (arrowhead, lane 4) that is eliminated when the putative Lef-1 site is deleted (lane 2). Small oligos containing the sequence CTTTGAA compete this shift (compare lanes 6 and 7; competing oligos were at ~500-fold excess). Mutant oligos with the sequence CTATGAC at the same concentration do not compete (compare lane 7 and 8). Presence (+) or absence (−) of extract is indicated at the top of each gel lane.