Mutation of the Tinman consensus site in the -2775/-2432 enhancer results in loss of enhancer activity in cardiac cells.

<p>(A-F)-2775/-2432 <i>Mef2-lacZ</i> embryos at stages 14 (A-C) and 16 (D-F). (A,D)Antibody stain against MEF2. MEF2 could be detected in all cells of the heart and throughout the somatic mesoderm. (B,E)Antibody stain against β-Galactosidase. Activity of the late stage enhancer was almost identical to that of <i>Mef2</i> expression. In (B), the enhancer is just becoming active in the heart cells, therefore a few cells lacked activity at this stage. By stage 16 (E) all cardiac cells showed ßGal accumulation. (C)Merge of (A) and (B); (F)Merge of (D) and (E). (G-L)Embryos carrying the -2775/-2432 <i>Mef2-lacZ</i> enhancer with the Tinman consensus site mutated, at stages 14 (G-I) or 16 (J-L). (G,J)Antibody stain against MEF2. MEF2 marks all cells of the heart and the somatic mesoderm. (H,K)Antibody stain against β-Galactosidase. Activity of the mutated enhancer was completely lost from the cardiac cells, and was slightly reduced in the somatic mesoderm. (I)Merge of (G) and (H). (L)Merge of (J) and (K). Arrows point to MEF2 positive cardiac cells, arrowheads point to the same cells lacking β-Galactosidase. (M-R) Embryos carrying the -2775/-2432 <i>Mef2-lacZ</i> enhancer with the three Pannier consensus sites mutated, at stages 14 (M-O) or 16 (P-R). (O)Merge of (M) and (N). (R) Merge of (P) and (Q). Arrows point to cardiac cells, arrow heads point to cells that have lost enhancer activity. Asterisk denotes activity in the amnioserosa. Bar, 100μm.