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Fig. 6. In vivo regulation of Serrate and Delta localization and stability by D-mib. In all discs excepting E and F, Delta is in red and Serrate in green. (A-D) Tests of endogenous D-mib function. Wing imaginal discs from wild-type (A,C) and D-mib1 (B,D). Apart from the defective wing pouch development, Dl expression is fairly normal in D-mib discs (B), but elevated levels of Serrate are present and localized primarily to the plasma membrane (D). Imaging of Delta and Serrate in A-D was performed identically, so that relative protein levels between discs is comparable. (E-P) Tests of ectopic D-mib function. In these experiments, D-mib isoforms are expressed in a stripe at the anteroposterior compartment boundary using dpp-Gal4. (E,F) dpp-Gal4, UAS-nGFP double stained for GFP (E) and GFP + Delta (F). The L3 wing vein expression of Delta is contained within the domain of dpp-Gal4 activity. (G-L) Wing discs double stained for Serrate and Delta. In all cases, the L3 wing vein domain is marked with an asterisk, and insets depict magnified apical views from this region. (G,H) Wild type. (I,J) dpp-Gal4, UAS-D-mib discs show a reduction of Serrate from the apical membrane and a strong decrease in both the apical and total level of Delta. (K,L) dpp-Gal4, UAS-D-mib{Delta}RF disc displays strongly increased levels of both Serrate and Delta. Endogenous margin-specific expression of Serrate and Delta is also interrupted (arrowhead). Note the large apical intracellular aggregates of Serrate (K, inset). (M-P) Effect of D-mib proteins on exogenous Delta. In these panels, one copy of hs-Delta is present, and animals were heat-shocked at 38°C for 40 minutes, then allowed to rest for the indicated period of time prior to dissection and fixation. The regions shown in (N-P) correspond to the boxed region in F. (M) hs-Delta/+, 40 minute rest. Delta is present at the plasma membrane of all cells. (N) hs-Delta, dpp-Gal4, UAS-D-mib, 40 minute rest. All Delta within the D-mib-expressing domain is vesicular. (O) hs-Delta, dpp-Gal4, UAS-D-mib, 90 minute rest. All Delta within the D-mib-expressing domain has been degraded. (P) hs-Delta, dpp-Gal4, UAS-D-mib{Delta}RF, 120 minute rest. D-mib{Delta}RF fails to efficiently induce either the internalization or degradation of Delta.





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