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Fig. 5. Ser is directly regulated by the Wg pathway. (A-E) The Ser-lacZ fusion gene (construct 8) is upregulated by activated Wg signaling. Arm^S10 is expressed in random clones (marked by green GFP in A,B,D). Ser-lacZ is visualized in blue (B) or in white (C). Dl (red), a known target gene of the Wg pathway, serves as a positive control and is detected by its antibody (E). Both Ser-lacZ (B,C) and Dl (E) are upregulated in Arm^S10 expressing cells, as indicated by arrows. The clone (arrowhead in B and D) located outside of the wing pouch has no effect on Ser-lacZ and Dl expression. (F-G) Ser-lacZ is downregulated by DN-dTCF. (F) Ser-lacZ expression in a wild-type background without en-Gal4 but with UAS-DN-dTCF. Expression is shown in the glowover mode (see legend for Fig. 4H,I); Ser-lacZ is expressed at higher levels dorsally. (G) UAS-DN-dTCF is expressed in the posterior compartment of the wing disc under the control of en-Gal4. Note that Ser-lacZ expression is eliminated in the ventral posterior compartment. The reduction of lacZ expression in the dorsal posterior compartment is significant, when compared with Ser-lacZ expression in a wild-type background. (H-K) DNase I footprinting analysis of the dTCF-HMG protein bound to the 794 bp Ser wing enhancer. Autoradiograms of denatured polyacrylamide gels show the separated products of DNase I digestion of dTCF-HMG/794 bp Ser wing enhancer complexes with relative amounts of dTCF-HMG protein (1x, about 2 µg protein; 3x and 6x, protein increased threefold and sixfold, respectively), or no dTCF-HMG (lanes `c' for control). The DNase I-sensitive bases protected by dTCF-HMG are marked, and their corresponding DNA sequences are shown (site A-site I). The DNA sequencing products of the 794 bp Ser wing enhancer are shown here with G (ddGTP) and A (ddATP), or C (ddCTP) and T (ddTTP), in the first two lanes. (L) Alignment of sequences that are bound by dTCF-HMG (from H-K). Sites A, F and I match the dTCF CCTTTGATCTT consensus, except for the unmatched nucleotides shown in red. Sites C, D, E and H are a good match for the HMG consensus, except for an unmatched guanine at site E. The non-canonical sequences at sites B and G show no obvious homology to either dTCF or HMG binding consensus sequences, except for a stretch of three thymidine residues in the middle. (M) Expression of the (mdTCF)Ser-lacZ transgene. The (mdTCF)Ser-lacZ construct contains mutations in all nine dTCF-binding elements. In the late third instar, (mdTCF)Ser-lacZ expression was greatly reduced in cells flanking the DV boundary (arrows), as compared to a wild-type Ser-lacZ disc (Fig. 3X4). Note that lacZ expression levels were higher in the notum (open arrowheads), where Ser expression is regulated independently of the Wg/dTCF pathway; lacZ expression in presumptive veins L3, L4 and L5 (arrowheads) was also detected.

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