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doi: 10.1242/10.1242/dev.00096

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Control of Drosophila eye specification by Wingless signalling

MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK

View larger version (117K): [in a new window]   Fig. 1. Activation of the Wingless signalling pathway blocks eye differentiation. (A-C) Scanning EM images of adult eyes with axin- clones. axin mutant cells induce the differentiation of frons cuticle corresponding to the dorsal head (arrowhead in B), but also naked cuticle (arrow in B). (C) Large axin mutant clone causing a tube-like overgrowth with multiple macrochaetae (arrow). (D-G) In third instar eye discs, axin mutant clones (which lack red anti-lacZ staining) occasionally express Distal-less (green); anterior towards the left. The expression of Distal-less is associated with tube-like overgrowth. These overgrowths appear in a different focal plane that can be seen in transverse sections of the disc (E-G; the positions of the sections are indicated by the white lines in D).

View larger version (51K): [in a new window]   Fig. 2. Excess proliferation in axin- mutant clones. (A) axin- mitotic clones were induced 60 hours after egg laying (AEL) using the GAL4/GAL80 system and eye/antennal discs were analysed by FACS at 108 hours AEL. Using this marker system, the mutant cells are positively marked by GFP. The black trace represents GFP-negative (wild-type) cells and red line represents GFP-positive (axin-) cells. The proportion of cells in G2 and S is increased in axin- cells, consistent with the hyper-proliferation phenotype observed in these clones. (B) An axin- Minute clone (marked by absence of red) stained with anti-BrdU (green). The focal plane shown is at the level of the clonal overgrowth so the S-phase associated with the second mitotic wave (just posterior to the morphogenetic furrow, arrowhead) is not seen.

View larger version (111K): [in a new window]   Fig. 3. Wg signalling regulates the expression of Eyes absent. axin- M+ clones were induced 60±12 (A-H) and 108±12 hours AEL (I-K). In all panels, Eyes absent protein is shown in green and mutant clones are indicated by absence of red anti-lacZ staining. (A,B) In axin mutant clones (surrounded by broken lines) the expression of Eyes absent is abolished in the region anterior to the morphogenetic furrow (approximate furrow positions are marked with arrows in B and I); note, however, that Eyes absent is present in cells in the posterior region of the eye discs (arrowhead). Eyes absent expression is also eliminated in small internal clones anterior to the furrow (arrow in A). (C-E) A transverse section in the anterior region of the disc shown in A (red line) confirms that Eyes absent expression is autonomously eliminated from the clone. (F-G) In a more posterior transverse section from the same disc (blue line), we observe that Eyes absent is expressed in mutant cells. The overgrowth of the mutant cell leads to the folded morphology of the tissue. (I-K) Small clones induced late in eye development also prevent the expression of Eyes absent. However, in some mutant cells close to the morphogenetic furrow (position indicated with arrows in I). Eyes absent is still present at low levels (arrow in J). Panels J and K show a different disc from I, at higher magnification.

View larger version (133K): [in a new window]   Fig. 5. axin- clones induced in a non-Minute background also cause overgrowth and loss of eye specification genes. In this case, mutant clones are positively marked in by GFP staining (in red, for consistency with other figures), using the Gal4/Gal80 system. (A) Eyes absent expression (green) is abolished in anterior but not posterior axin- clones. Red box: transverse section of a small anterior clone indicated by red line; mutant cells have grown out of the plane of the epithelium and do not express Eyes absent. Blue box: transverse section of a posterior clone indicated by blue line; mutant cells show extensive overgrowth (arrow) and some express Eyes absent. (B,C) Dachshund expression (green) is autonomously abolished in anterior axin- clones (e.g. arrowhead in C). (D,E) Sine oculis expression is autonomously abolished in anterior axin- clones (e.g. arrowhead in E). (D,E) Projections of several confocal sections; to demonstrate the autonomy of this loss, the inset in E shows a single confocal section of the clones indicated by a box in D (the central cells in the clone are not seen as they have grown out of the plane of the section).

View larger version (60K): [in a new window]   Fig. 6. Eyes absent and sine oculis have complementary expression patterns to Wingless. (A-C) Expression of wg-lacZ (green) and Eyes absent protein (red and white in right-hand panels) during third instar eye disc development. (A) In the early third instar eye disc, before initiation of the morphogenetic furrow, Wingless is expressed in the anterior lateral margin of the eye disc, whereas Eyes absent is expressed throughout the eye primordia. (B) In the mid-third instar eye disc, when the morphogenetic furrow has started its posterior to anterior progression, the expression pattern of both genes remains similar. (C) A basal view (single confocal section) of a late third instar eye disc, showing that Eyes absent is missing from the anterior lateral margin where Wingless is expressed. Note that in posterior regions some overlap is seen (indicated by yellow staining). (D) Expression of sine oculis-lacZ (red, white in right-hand panels) and Wingless protein is precisely complementary in the anterior region, but slightly overlap in the posterior lateral region (single confocal section).

View larger version (79K): [in a new window]   Fig. 4. Clones of arm*-expressing cells (red) abolish the expression of sine oculis-lacZ (A), Dachshund protein (B) and Eyes absent protein (C) (all shown in green and white in right-hand panel). As observed in axin- mutant clones, the expression of these genes is eliminated in anterior clones (arrows in B and C), but is often ectopically expressed (arrowhead in C) or not eliminated (arrowhead in B) in posterior clones.

View larger version (141K): [in a new window]   Fig. 7. Wingless signalling is necessary to prevent the expression of Eyes absent and Dachshund in the anterolateral margin of the eye disc. (A,B) Eyes absent (green) is ectopically expressed (arrows) in a fz1 fz2 mutant clone (lack of red staining); B shows a higher magnification of the relevant region of A. (C) A fz1 fz2 mutant clone in a similar position to the one shown in A ectopically expresses Dachshund (green).

View larger version (146K): [in a new window]   Fig. 8. Ectopic expression of eyes absent partially rescues expression of Sine oculis and Dachshund in axin- clones. axin- clones that simultaneously express eyes absent (using the Gal4/Gal80 system) are shown in red. (A) Dachshund expression (green) is partially rescued (arrowhead) in some, but not all (arrow), of these clones (compare with Fig. 5B,C). (B) Sine oculis expression is also partially rescued in these clones (arrowhead); compare with Fig. 5D,E. The inset shows a clone in the antennal region of the disc with ectopic expression of Sine oculis (in green).