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First published online May 28, 2004
doi: 10.1242/10.1242/dev.01177

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Nemo is an inducible antagonist of Wingless signaling during Drosophila wing development

Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, V5A 1S6, Canada

View larger version (111K): [in a new window]   Fig. 2. nmo antagonizes Wg signaling during wing development. (A) A wild-type adult wing. (B) sd-Gal4/UAS-nmo. (C) zw3m11-1/+; sd-Gal4/UAS-nmo. (D) The null allele nmoDB24. (E) sd-Gal4/UAS-DFz2N. (F) Loss of nmo in sd-Gal4/UAS-Fz2N; nmoDB24/nmoDB24 flies rescues the severe wing defect seen in E. (G) sd-Gal4/+; UAS-Daxin causes a wing-to-notum transformation (see inset). (H,I) Reductions in nmo rescue in a dose-dependent manner in (H) sd-Gal4/+; UAS-Daxin, nmoDB24/+ and (I) sd-Gal4/+; UAS-Daxin, nmoDB24/nmoDB24. (J) ap-Gal4/+; UAS-Daxin. Reductions in nmo rescue this phenotype in a dose-dependent manner. (K) ap-Gal4/+; UAS-Daxin, nmoDB24/+ and (L) ap-Gal4/+; UAS-Daxin, nmoDB24/nmoDB24. (M) 71B>fluarm. (N) 71B>nmo. (O) UAS-fluarm/UAS-nmo; 71B-Gal4/+.

View larger version (78K): [in a new window]   Fig. 1. nmo expression in the wing imaginal disc was examined in nmo-lacZ. (A) In second instar discs, weak nmo expression is seen at the periphery of the future wing pouch (arrow). (B) In early third instar discs, low level expression is at the DV boundary (arrow) and encircling the wing pouch. (C) In late third instar, high levels of nmo expression are seen in two stripes flanking the DV boundary and in a ring around the pouch. nmo is also seen in the L3, L4 and L5 vein primordia (arrowheads) and in several spots in the presumptive notum (arrow). (D) In situ hybridization using an antisense nmo RNA probe. (E-G) Co-localization with Wg. Discs were double stained with (E) anti-ß-gal and (G) anti-Wg antibodies and the images were merged to show overlap (F). Wing imaginal discs are orientated anterior towards the left, dorsal side upwards.

View larger version (71K): [in a new window]   Fig. 3. Nemo plays a role in specification of macrochaete bristles on the adult notum. (A) A wild-type notum. (B) ap>nmo flies show loss of macrochaetes on the notum. A few scuttelar bristles remain (arrows). (C) zw3m11-1/+; ap>nmo. (D) Ectopic expression of Daxin enhances the phenotype in ap-Gal4, UAS-nmo/+; UAS-Daxin/+ flies. (E) ap>nmo wing. (F) zw3m11-1/+; ap>nmo wing.

View larger version (74K): [in a new window]   Fig. 4. Both reduction of nmo and ectopic nmo can affect Wg-dependent gene expression. (A) Dll-lacZ expression is seen in a broad domain centered on the DV boundary with areas of increased expression at the anterior and posterior edges of the DV boundary. (B) Ectopic Nemo in ap>nmo can greatly reduce the Dll-lacZ expression, particularly in the posterior margin region. Dll expression is enhanced in nmo mutant clones. (C,D) nmoDB24 clones (marked by the absence of GFP, green). (D,E) Expression of Dll-lacZ (anti ß-gal, red) is also increased in nmoDB24 clones (arrow in E).

View larger version (101K): [in a new window]   Fig. 5. Neither reduction of nmo nor ectopic nmo can affect Wg expression. Both somatic nmoDB24 clones (A,B; marked by the absence of GFP, green) and flip-out clones ectopically expressing UAS-nmo (D,E; marked by the areas of brighter GFP staining, green) were induced. The discs were stained for Wg protein to determine whether modulation of nmo could affect the Wg expression pattern (anti-Wg antibody, red in B,C,E,F). Anti-Wg stain reveals a wild-type pattern in both reduced and ectopic Nemo.

View larger version (80K): [in a new window]   Fig. 6. Wg signaling positively regulates the expression of nmo. (A) vg-Gal4 is expressed along the DV boundary in vg>lacZ. (B) nmo expression in vg-Gal4/UAS-fluarm; nmo-lacZ/+ mid third instar larval discs is greatly expanded, especially in the posterior periphery. (C) dpp>fluarm causes ectopic nmo expression along the AP boundary. (D-F) nmo-lacZ expression (E,F; anti ß-gal, red) in fluarm flip-out clones (D,E; marked by the areas of brighter GFP staining). (G-I) Flip-out clones ectopically expressing UAS-Daxin (G,H) also result in decreased nmo expression (H,I). (J-L) In dshv26 somatic clones (J,K; marked by the absence of GFP, green) nmo-lacZ expression is reduced cell autonomously (K,L; anti-ß-gal, red).

View larger version (83K): [in a new window]   Fig. 7. Nemo can influence Arm stabilization. (A,B) nmo gene expression (as monitored by nmo-lacZ, anti ß-gal, red) overlaps with stabilized Arm protein (B,C; anti-Arm, green) in third instar discs. There are distinct regions in which higher nmo expression in A excludes high levels of Arm (arrow in C) and in which high levels of Arm seen in C coincide with reduced nmo (arrowhead in A). In flip-out clones ectopically expressing Nemo (D,E; marked by the areas of brighter GFP staining), the stabilization of Arm protein appears reduced in a cell-autonomous manner (E,F; anti-Arm, red; arrows in F).

View larger version (19K): [in a new window]   Fig. 8. The role of negative feedback inhibitors in Wg signaling. Drosophila Wg signaling is controlled by a number of induced inhibitors including Nkd and Wf. We show that Wg also regulates Nemo expression and that Nemo in turn can antagonize Wg during wing patterning.