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Different mechanisms initiate and maintain wingless expression in the Drosophila wing hinge

¹ Centro de Biología Molecular-Severo Ochoa/C.S.I.C., Facultad de Ciencias-CV, Universidad Autónoma-Cantoblanco, 28049 Madrid, Spain
² School of Biological Sciences, University of Sussex, Falmer, Brighton BN1 9QG, UK

View larger version (70K): [in a new window]   Fig. 1. wg expression and phenotypes of wild-type and mutant wing hinges. (A) Adult wing and third instar larval wing imaginal disc showing wg expression detected by antibody staining. Bars indicate the regions amplified in B-E. (B) wg expression detected by X-gal staining in adult wing and by anti-Wg antibody in the wing pouch. Red arrows indicate the inner ring (IR); black arrows indicate the corresponding region in the adult wing. The arrowhead indicates the outer ring (OR). (C-E) Mutant phenotypes of spdfg (C), rn2–2 (D) and nub2 (E). In all cases the hinge is deleted and the expression of wg, which is detected by staining of antibody to Wg, in the IR is missing.

View larger version (73K): [in a new window]   Fig. 2. vg, rn and nub expression patterns and their relationship to the IR. Wild-type wing disc from late second (A-C), early third (D) and late third (E-G) instar larvae. (H-J) spdfg mature wing discs. In late second instar larvae vg, rn and nub expression domains are almost coincident (A-B), and wg is expressed in a stripe that delimits the presumptive wing margin (C). In early third instar larvae, wg is also expressed in a ring of cells that do not express vg (D, white arrow). (E-G) In mature wing discs vg, rn and nub are expressed in three concentric domains (arrows): vg and wg (E), rn and wg (F), and nub and wg (G). (H-J) spd-lacZ, vg, rn and nub expression patterns in spdfg discs. Note that the IR co-expresses with rn (I) and nub (J), but abuts the vg domain (H). Because of different focal planes it is not possible to merge the different channels over the whole of the Wg domain at this magnification. (K) Summary of patterns of gene expression. The mature wing hinge has five characteristic folds (arrows) that coincide with domains of gene expression. Vertical bars represent the domains of expression of nub, rn, vg and wg in the IR and the OR. Note that although domain 2 includes the IR, only the cells in the proximal-most sector express wg. In all discs, anterior is leftwards and the dorsal notum is upwards. The wing margin, identified by wg expression, is considered the distal-most region of the wing.

View larger version (115K): [in a new window]   Fig. 3. vg is required to activate the expression of wg, rn and nub in the wing pouch. (A-C) vg83b27R. The expression of wg (A), rn (B) and nub (C) in the wing pouch is missing (arrows; see Fig. 1F,G for wild-type expression). The expression in notum and legs was not affected. Ectopic expression of vg is sufficient to activate rn and nub. (D-F) dpp-GAL4/UAS-vg. Patterns of expression of wg (D), rn (E) and nub (F) in the eye/antenna disc. Arrowheads indicate the dpp expression domain in the antenna. rn and nub are expressed in similar patterns in leg and antenna. In D-F, ventral is leftwards.

View larger version (111K): [in a new window]   Fig. 4. vg and rn mediate the activation of the IR. (A-F) Clones of vg-expressing cells. Clone cells are labelled green (GFP), Wg is labelled in blue (wg antibody) and Rn in red (rn-lacZ). When we examine the expression of wg in these clones we found three distinct results: (1) wg is expressed in a narrow ring of cells that abuts the clone border (A); (2) wg is expressed in a broader ring of cells that still abuts the clone border (B); and (3) wg is expressed in a ring that stands several cells away from the clone border (C). We infer that these three distinct results represent three stages and reproduce the process of activation of the IR throughout normal development. (D-F) vg-expressing clones activate rn expression within the cells of the clone and in surrounding cells. This activation is restricted to the Nub domain. (D) Non-autonomous activation of rn in a vg-expressing clone. The arrow indicates the fold that delimits the expression of nub. (E) Two clones out of the Nub domain (arrowheads) and a clone within the Nub domain (arrow). Owing to different planes of focus, not all the cells of the clone show rn expression. (F) vg-expressing clones that do not activate rn expression in cells out of the clone do not express wg (arrowhead). (G,H) Clones of rn-expressing cells: clone cells are red (GFP) and Wg is blue. Rn autonomously activates wg expression, but only in the Nub domain and in cells that are close to the Vg domain. (I) wg expression in ap-GAL4/UAS-vg wing disc. Note the lack of dorsal wg expression (arrow).

View larger version (94K): [in a new window]   Fig. 5. Rn and Nub are required for wg expression in the IR. (A-B) rn2-2 clones, revealed by the absence of GFP (green), cell autonomously remove wg expression (red) in the IR. Other domains of wg expressions are not affected. (C,D) nub1 clones, revealed by the absence of GFP (green), also remove wg expression (red) in the IR. As it has previously been reported, wg expression in the wing margin is expanded in these clones (Neumann and Cohen, 1998). Arrows indicate the IR. wg expression is detected with antibody to Wg. Dorsal is upwards and anterior is leftwards.

View larger version (91K): [in a new window]   Fig. 6. Cell interactions at the boundary of vg-expressing cells drive the expression of the IR enhancer. (A-G) dpp-GAL4/UAS-hth removes vg expression (A), but does not affect expression of rn (B) or nub (C) (arrows). Note that the activation of the N-dependent vg BE is not repressed (arrowheads in A). wg is ectopically expressed within the Dpp domain in two stripes of cells that abut vg-expressing cells (D-F, arrows). In second instar larvae, the two stripes of Wg are more apparent (D). In third instar larvae, only one stripe is seen because of a fold in the epithelium (F). (G,H) Two results indicate that the new Wg stripe corresponds to the activation of the IR enhancer: Dll expression is missing (G); and n Nts; dpp-GAL4/UAS-hth (H), wg expression in the wing margin is missing (arrowheads) but the IR and the new stripe of wg is not affected (red arrow). (I) hth is unable to directly activate wg expression: in en-GAL4/UAS-hth only posterior cells in the AP boundary activate the IR (red arrow), indicating that cell interactions with vg-expressing cells are required.

View larger version (105K): [in a new window]   Fig. 7. (A,B) hth expression detected with antibody to ß-galactosidase in hth-lacZ wing discs. hth is expressed in the wing hinge in two rings (A). The inner ring (red arrow) and the outer ring (red arrowhead) overlap with the IR and the OR of Wg. (B) In spdfg discs, the hth expression in the inner ring is missing but the outer ring is not affected. (C) rn lineage tracing. Expression of rn visualised by rn-lacZ (red) and by rn-GAL4/UAS-FLP Act>>GAL4/UAS-GFP (green), which labels all the cells that expressed rn at any time during the development of the disc. Note that the green channel shows a broader domain, indicating cells that have lost rn expression. This result suggests that in the border of Rn domain, cells have a tendency to lose rn expression. (D,E) hthP2 clones, revealed by the absence of GFP (green), remove wg expression (red) in the IR (white arrows) when observed in mature wing discs (D), but do not do so when observed in earlier discs (E). This indicates that the maintenance, but not the initiation, of wg expression requires Hth function. (F-H) wg expression in the IR is required to maintain sharp borders in the vg expression domain. Expression of vg QE-lacZ in wild-type (F), spdfg (G) and Nts (H) [at restrictive temperature (30°C)] wing discs detected with antibody to ß-galactosidase. The border of the Vg domain (black arrows) is sharp in wild-type larvae or when wg is removed from the wing margin (H), but less well defined when wg is removed from the IR (G).

View larger version (28K): [in a new window]   Fig. 8. A model for the development of the wing hinge. The figure represents the evolution of gene expression during development in cells of the dorsal wing hinge. In second instar larvae vg (red) and tsh (grey) are expressed in complementary patterns (1). The vg-expressing cells correspond to the presumptive wing pouch. A signal coming from the vg-expressing cells activates nub and rn expression in slightly broader domains (2). In early third instar larvae, cells expressing rn and nub but not vg (green) are competent to activate the wg IR enhancer (yellow) when induced by a signal coming from the vg-expressing cells (3). wg expression stimulates local cell proliferation that expands the different domains and moves the IR domain several cells away from vg-expressing cells (4). At this time, Wg signalling activates hth expression in the IR (orange). The combination of both Wg-dependent Hth and Vg-dependent Rn activates a Vg-independent mechanism that maintains wg expression in the IR. As soon as local cell proliferation moves rn-expressing cells away from the Vg domain, they lose rn expression, and consequently also lose wg and hth expression. Thus, wg expression in the IR is maintained at the border of the Rn domain, which proximally restricts the IR domain. In this process three new domains have been generated by local cell interactions that do not involve any cell lineage restriction. Note that the cells that belong to the new domain of rn-expressing cells between the IR and the Vg domain (green cells in 4) loses the ability to activate wg by a mechanism that, we propose, involves a Vg-dependent repressor. Therefore, the IR domain is proximally and distally restricted.