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. 2012 Apr 15;139(8):1457–1466. doi: 10.1242/dev.069005

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© 2012.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Share Alike License (http://creativecommons.org/licenses/by-nc-sa/3.0), which permits unrestricted non-commercial use, distribution and reproduction in any medium provided that the original work is properly cited and all further distributions of the work or adaptation are subject to the same Creative Commons License terms.

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Fig. 2. — Expression driven by the Ndg enhancer in the gut musculature is significantly reduced by mutating either the Fkh1 site or both Fkh2 and Fkh3 sites simultaneously. (A-F″) The expression of GFP reporters for the wild-type (A-A″) and mutated (B-F″) Ndg enhancers was compared at stage 16. Expression of a β-galactosidase reporter from the wild-type Ndg enhancer, Ndg^WT-lacZ, was used in every embryo as a reference and an internal control. Anterior is towards the left in every panel. Mutating either the Fkh2 (B-B″) or the Fkh3 (C-C″) site alone had no significant effect on reporter expression in the gut musculature, while mutating both these sites simultaneously (D-D″) resulted in a dramatic decrease in visceral muscle expression. Mutating only the Fkh1-binding site (E-E″) also caused a significant decrease in Ndg reporter expression, while mutating all three sites simultaneously (F-F″) resulted in the complete inactivation of GFP reporter expression in the visceral muscle. Note that although Ndg expression in the heart (arrows) is visible in every embryo, it is more readily apparent for the GFP reporters, which is a consequence of: (1) GFP being expressed throughout the entire cytoplasm, as opposed to β-galactosidase, which is confined to the much smaller nuclei; (2) the plane of focus being on the gut muscle and not the heart; and (3) de-repression into additional cardiac cells occurring only for the GFP reporters in which Fkh sites are mutated in B-F″ (also see Fig. 4 and the main text).