Fig. 3. Ser is directly regulated by Apterous (Ap). (A-I) The Ser-lacZ fusion gene (construct 10) is upregulated by Ap. (A,B) Ser-lacZ (red) is expressed in a stripe in the dorsal compartment around 24 hours after the L2/L3 molt in third instar in a wild-type background. (A,C) UAS-GFP (green) under dpp-Gal4 control reveals the wild-type dpp expression pattern at the AP border. ChAp is expressed under dpp-Gal4, which induces ectopic Ser-lacZ expression more ventrally along AP border in early third instar (arrows; D,E) and late third instar (arrows; G,H), as compared with the same stage discs in a wild-type background (B,Q). The ChAp expressing cells are marked by co-overexpression of GFP (green) under dpp-Gal4 (D,F,G,I). (J-Q) Ser-lacZ (construct 10) is downregulated by dLMO. UAS-dLMO and UAS-GFP (green, to mark dLMO expressing cells) are co-expressed at the AP border under ptc-Gal4. Note that ptc-Gal4 has a stronger expression level more posteriorly. Ser-lacZ expression is downregulated in dLMO expressing cells, particularly in the posterior compartment in early third instar (arrows; J,K). The reduction of Ser-lacZ in dLMO expressing cells is less dramatic in late third instar (N,O). The expression patterns of Ser-lacZ in a wild-type background are also shown for comparison (M, early third instar; Q, late third instar). (R-W) Binding of the Ap homeodomain (ApLIM, 6xHIS-tagged) to the 794 bp Ser minimal wing enhancer is direct and sequence specific. (R-V) DNase I footprinting analysis of the Ap homeodomain bound to the 794 bp Ser wing enhancer. Autoradiograms of denaturing polyacrylamide gels show the separated products after DNase I digestion of ApLIM/794 bp Ser wing enhancer complexes, and the relative amounts of ApLIM protein (1x, 20ng protein; 3x, protein increased threefold) or no ApLIM protein (lanes `c' for control). The DNase I-sensitive sequences protected by ApLIM are marked (site A-site N). 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. (W) Alignment of sequences that bind ApLIM (from R-V). The sequences of sites H and I do not match either the TAATNN or the CAATNN consensus and are shown with non-matched nucleotides in red; they are determined arbitrarily. rc, reverse complementary sequence. (X1-Y4) X-Gal staining to reveal in vivo activity of the wild-type Ser-lacZ (construct 10; X1-X4) and (mAp)Ser-lacZ transgenes (Y1-Y4). The (mAp)Ser-lacZ construct contains mutations in all fourteen Ap-binding elements. Ser-lacZ expression was detected in dorsal cells of wing and haltere discs at 7.5 hours after the L2/L3 molt (X1). At 24 hours after the L2/L3 molt, expression was restricted to a stripe in the dorsal compartment of the wing disc, and in the entire dorsal compartment of the haltere disc (X2). At the corresponding stages, (mAp)Ser-lacZ displayed no enhancer activity in the wing and haltere discs (Y1,Y2). By 36 hours after the L2/L3 molt, in mid third instar, Ser-lacZ was expressed along the DV border, and at a low level in the ventral compartment of the wing disc (arrow) (X3); (mAp)Ser-lacZ expression was much reduced (arrow) in the wing disc and was not detected in the haltere disc (Y3). At the end of third instar, Ser-lacZ expression in the wing and haltere discs was evident (X4); (mAp)Ser-lacZ expression was reduced and restricted (arrows; Y4). Note that in Y1, dorsal is to the left for the wing disc. The insets in the upper right hand corner of (X1-3,Y3), and in the lower right hand corner of (X3,Y3) show the wing and haltere discs, at lower magnification.