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doi: 10.1242/10.1242/dev.00393

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Regulation of Wingless and Vestigial expression in wing and haltere discs of Drosophila

Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad, 500 007, India

View larger version (76K): [in a new window]   Fig. 1. Wg is required for the maintenance of Vg expression in the DV boundary. (A) Wild-type expression pattern of Vg in wing discs. Vg is expressed in both DV and non-DV cells. (B,C) Vg expression in DV and non-DV cells is regulated by vg-BE (B) and vg-QE (C), respectively. (D-F) vg-GAL4/UAS-DN-TCF/pan wing discs stained with anti-Vg antibodies (D), vg-BE (E) and vg-QE (F). Misexpression of DN-TCF/pan in wing discs downregulates Vg expression in both DV and non-DV cells. (G) Wing disc with armH8.6/armH8.6 mitotic clones. (G1) lacZ marker (arm-lacZ P[FRT]18A); loss of lacZ expression marks armH8.6/armH8.6 cells. (G2) Vg expression pattern in the same disc. (G3) DAPI staining in the same disc. (G4) Merge image of G1-G3. Note that in a representative armH8.6/armH8.6 clone at the DV boundary (arrow), Vg expression is downregulated. Downregulation of Vg in non-DV cells (arrowhead) in arm– clones is also shown.

View larger version (113K): [in a new window]   Fig. 2. Autoregulation of Vg in non-DV cells of wing discs. (A1-3) Wing disc with vg1/vg1 clones. (A1) Myc marker (P[FRT]42 Myc); loss of Myc marks vg1/vg1 cells. (A2) Expression pattern of vg-QE in the same disc. (A3) DAPI staining in the same disc. (A4) Merged image of A1-A3. Note the representative vg1/vg1 clones in non-DV cells (arrows), which are positive for DAPI, indicating the survival of the clones. vg-QE is not expressed in these clones, which provides genetic evidence for Vg autoregulation. (B-C) vg1/vg1 wing discs stained with anti-Vg antibodies and DAPI (B), and anti-Wg antibodies (C). Note no Vg expression is seen in vg1/vg1 wing discs. Wg expression at the DV boundary is also absent. (D,E) Wild-type (D) and vg1/vg1 (E) wing discs showing the expression pattern of N23-GAL4. The discs are also stained with DAPI. Note that N23-GAL4, which is expressed only in non-DV cells, still shows separation of dorsal and ventral compartments at the presumptive DV boundary of vg1/vg1 discs. Thus, loss of Wg seen in vg1/vg1 wing discs is not caused by loss of the DV boundary per se. (F) N23-GAL4/UAS-Vg wing discs stained for vg-QE (red) and Wg (green). Note activation of both vg-QE and Wg (arrow) outside the wing pouch. In all such cases, Wg expression always surrounded but did not overlap vg-QE expression [similar observations have been made by Liu et al. (Liu et al., 2000)]. (G) vg1/vg1; N23-GAL4/UAS-Vg stained for vg-QE (red) and Wg (green). Note the very high levels of vg-QE activity in the pouch. No Wg expression was seen in the DV boundary, suggesting that autoregulation of Vg through its quadrant enhancer is independent of Wg. Note also that the size of the wing pouch is nearly normal. (H) vg1/vg1 adult wing blade. (I) vg1/vg1; N23-GAL4/UAS-Vg adult wing blade showing partial rescue of vg1 phenotype.

View larger version (85K): [in a new window]   Fig. 3. Wg signaling is required, but is not sufficient to activate vg-QE. (A-B) Wild-type wing disc showing the expression pattern of Vg (A) and dpp-GAL4 driver (B). (C,D) dpp-GAL4/UAS-Nintra-wing discs stained for Vg (C) and Wg (D). Activation of Vg by ectopic Nintra expression is non-cell autonomous whereas that of Wg is cell autonomous. (E) Dpp-GAL4/UAS-activated Arm showing cell-autonomous activation of Vg. (F1-4) Wing disc with armH8.6/armH8.6 mitotic clones. (F1) GFP marker (Ubi-GFP P[FRT]18A), (F2) vg-QE and (F3) DAPI staining. (F4) Merged image of F1-F3. Loss of GFP expression marks armH8.6/armH8.6 cells. vg-QE is not expressed in these clones.

View larger version (136K): [in a new window]   Fig. 4. Enhanced degradation of Arm in haltere discs. (A-D) Wild-type expression of Arm in wing (A,B) and haltere (C,D) discs. (A1,C1) Higher magnification images of wing (A1) and haltere pouch (C1). In wing discs, cells surrounding Wg-expressing DV boundary cells show higher levels of Arm (A,A1,B). In haltere discs, levels of Arm at the DV boundary are indistinguishable from those of non-DV cells (C,C1,D). In a few haltere discs, we observed somewhat higher levels of Arm in the cells that intersect the A/P and DV boundaries (D). (E) vg-GAL4/UAS-Ubx-wing disc. Ectopic Ubx downregulates Arm levels in the DV cells of the wing disc. (F,G) omb-GAL4/UAS-armS10-wing (F) and -haltere (G) discs. Wing and haltere discs show comparable levels of degradation-resistant Arm expressed from UAS-armS10. (H,I) omb-GAL4/UAS-armS2-wing (H) and -haltere (I) discs. In wing discs, overexpression of wild-type Arm from UAS-armS2 leads to accumulation of Arm only in the presumptive DV boundary and hinge cells. However, no significant accumulation of wild-type Arm (from armS2) is seen in the DV boundary of haltere discs, although, as in wing discs, hinge cells accumulate large amounts of wild-type Arm. This suggests that Ubx enhances degradation of Arm in the haltere pouch.

View larger version (115K): [in a new window]   Fig. 5. Ubx-mediated inhibition of Arm stabilization is downstream of Sgg function. All discs in this figure are stained with anti-Arm antibodies. (A) vg-GAL4/UAS-Dsh-haltere disc. Overexpression of Dsh does not enhance Arm levels at the haltere DV boundary, suggesting that Ubx functions downstream of Dsh. (B,C) vg-GAL4/UAS-APC/CBD-wing (B) and -haltere (C) discs. Misexpression of human APC sequesters Arm only in cells where Sgg is inactive (Bhandari and Shashidhara, 2001): for example, at the DV boundary of the wing disc (B). In haltere discs, APC sequesters Arm only in the anterior compartment (C). This suggests that Sgg is inactive in the anterior compartment and active in the posterior compartment. (D) vg-GAL4/UAS-Dsh; UAS-APC/CBD-haltere disc. Overexpression of both Dsh and APC together causes sequestration of Arm in both anterior and posterior compartments. This is owing to the Dsh-mediated inhibition of Sgg activity followed by APC-mediated sequestration of Arm.

View larger version (45K): [in a new window]   Fig. 6. Differential regulation of Wg and Vg in wing and haltere discs. (A,B) vg-GAL4/UAS-DN-TCF/pan-wing (A) and -haltere (B) discs stained with anti-Wg antibodies. Misexpression of DN-TCF/pan downregulates Wg expression at the wing disc DV boundary, but not in haltere discs. (C,D) vg1/vg1 haltere discs stained for Vg (C) and Wg (D). Note the absence of Wg expression at the DV boundary, which suggests that Wg expression in haltere discs is dependent on Vg. (E) vg-GAL4/UAS-DN-TCF/pan-haltere disc stained for Vg. DN-TCF/pan does not have any affect on Vg expression, which suggests that its expression at the haltere DV boundary, unlike in wing discs, is independent of Wg signaling.

View larger version (101K): [in a new window]   Fig. 7. Ubx-mediated repression of Vg in non-DV cells is downstream to Arm and upstream to Vg-autoregulation. (A,B) dpp-GAL4/UAS-Nintra-haltere discs stained for Vg (A) and Wg (B). Unlike in wing discs, activation of Vg by ectopic Nintra is cell autonomous and Wg is not activated in haltere discs. (C,D) dpp-GAL4/UAS-activated Arm-haltere discs stained for Vg (C) and Wg (D). No activation of Vg and Wg was observed. Compare this with Fig. 3E, which shows cell-autonomous activation of Vg in wing discs by ectopic Arm. (E,F) Wild-type (E) and vg1/vg1- (F) haltere discs showing the expression pattern of N23-GAL4. As in wing discs, the GAL4 driver is expressed in non-DV cells of haltere discs, but only in the posterior compartment. (G,H) N23-GAL4/UAS-vg- (G) and vg1/vg1; N23-GAL4/UAS-vg- (H) haltere discs stained for vg-QE (red) and Wg (green). Note that Vg is capable of activating its quadrant enhancer in both wild-type and vg1/vg1 backgrounds. This suggests that downregulation of Vg by Ubx in non-DV cells in wild-type haltere discs is upstream of Vg-autoregulation.

View larger version (36K): [in a new window]   Fig. 8. Haltere-to-wing homeotic transformations induced by ectopic Vg. (A) Wild-type haltere. (B) omb-GAL4; UAS-vg haltere showing significant transformation of haltere capitellum to wing blade. Note the wing-like trichomes, which are larger, flatter and more pigmented and sparsely arranged than capitellum cells. (C) Ubx–/Ubx+ halteres showing mild haltere-to-wing transformation. This is generally marked by the appearance of one or two wing-margin bristles. (D) omb-GAL4; UAS-vg/Ubx– haltere showing enhanced homeotic transformation in a Ubx heterozygous background. The increase in the number of margin-bristles could be caused by the additive effects of increased growth, upregulated Wg signaling by overexpressed Vg and sensitized genetic (Ubx–/+) background. This further confirms that Vg is required for the correct interpretation of Wg signaling (Klein and Martinez-Arias, 1999). (E,F) Wild-type (E) and omb-GAL4; UAS-vg- (F) haltere discs stained with anti-Salm antibodies. Salm is not normally expressed in the haltere pouch (E), nor is it induced by ectopic Vg (F). (G) Wg and Vg regulation in wing and haltere discs. Figure shows how DV signals activate Vg in non-DV cells in wing discs, and the events that are downregulated by Ubx in haltere discs. Regulatory elements of Vg are represented in two boxes: green box, vg-quadrant enhancer; white box, other enhancers of Vg that respond to Wg and probably one more, hitherto unknown, DV signal. Once activated, Vg maintains its expression by autoregulation, which is mediated through its quadrant enhancer. The discontinuous lines shown for haltere discs are the steps inhibited by Ubx during haltere specification. At the top of the hierarchy, Ubx downregulates Wg expression at the DV boundary of the posterior compartment (not shown). Although Vg-autoregulation per se is not affected, in the absence of initial activation of Vg by Wg signaling vg-QE is not activated in haltere discs.