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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 (Ap LIM,
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.
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