Fig. 6. Requirement of Mad and Creb proteins for XC enhancer activity. Enhancer activity is visualized by in situ hybridization to lacZ transcripts. All panels show magnifications of lateral views of the midgut of stage 14 embryos. Arrowheads indicate the site of wg expression in PS8. Arrows indicate ectopic enhancer activity. All embryos have been processed under the same conditions and the staining times were identical. (A-C) Requirements in trans. (A) Wild-type embryo carrying the XC enhancer. The activity of the XC enhancer is lost in mad¹²-homozygous mutants (B) and is strongly reduced upon expression of a dominant-negative form of Drosophila CrebB in the mesoderm of 24B-Gal4/UAS-Creb(DN) embryos (C). (D-F) Requirements in cis. Mutation of the three DRS in XC(DRS1-2-3) results in the complete loss of enhancer activity in VM PS8 (D). It also induces ectopic activity (arrows) of the enhancer in endodermal cells from the central part of the midgut, and in a more anterior region close to the foregut/midgut boundary (D). XC(Creb1-2) that bears mutations in the two Creb binding sites shows a severely reduced enhancer activity (E). The mutation of the three DRSs and the two Creb binding sites results in a complete loss of enhancer activity (F), including in the territories where the enhancer is ectopically induced by XC(DRS1-2-3).