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First published online 13 August 2008
doi: 10.1242/dev.020800

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Reciprocal roles for bowl and lines in specifying the peripodial epithelium and the disc proper of the Drosophila wing primordium

¹ Program in Cell, Molecular and Developmental Biology, 150 Harrison Avenue, Boston, MA 02111, USA.
² Program in Genetics, Tufts University School of Medicine, Department of Anatomy and Cellular Biology, 150 Harrison Avenue, Boston, MA 02111, USA.

View larger version (69K): [in this window] [in a new window]   Fig. 1. Bowl accumulates in the PE and is absent from the DP. Spatiotemporal expression pattern of Bowl (red), wg-lacZ (green) or odd-lacZ (green). Optical sections at the plane of the PE (A,C,E) and DP (B,D,F). (A,B) 48 hours AEL; (C,D) 72 hours AEL; (E,F) 96 hours AEL. wg-lacZ was detected in the DP in a wedge of ventral cells (arrow in B). wg was upregulated along the DV compartment boundary (arrows in D and F). Bowl accumulated broadly in the PE (A,C,E) and was absent from the DP (B,D,F), except for the lateral margins of the notum (arrowheads in D and F). (G,H) 120-144 hours AEL; Bowl accumulated in the anterior margin of the PE (asterisks in G and H) and the lateral margins of the notum (arrowheads in H). However, Bowl was downregulated in the medial and posterior regions of the PE. (I) Everting discs; Bowl accumulated in the ventro-anterior hinge (asterisk) and in the lateral margins of the notum (not shown in this image). (J) odd-lacZ was detected in a similar pattern at this stage (arrowheads indicate the lateral margins of the notum, asterisks the ventro-anterior hinge). (K) Adult wings; odd-lacZ was detected in ventro-proximal hinge nuclei. (L) A schematic diagram illustrating the regulatory interactions linking drm, lines and bowl.

View larger version (115K): [in this window] [in a new window]   Fig. 2. The Lines and Bowl proteins accumulate in a reciprocal pattern across the PE and the DP. Clones expressing (A-C) Myc-Lines (weak insertion), (D) Flag-Bowl, (F) Drm or (G) Lines (strong insertion) were marked with nuclear GFP reporter, which accumulates to high levels in nuclei and to lower levels in the cytoplasm. (E) lines mutant clones were GFP-negative. Arrows in A-C,E-G indicate magnified areas shown in insets. (A-B'') Optical sections at the plane of the PE; Myc-Lines was enriched in the cytoplasm in the lateral margins of the PE (A-A''), and in the simple squamous PE (B-B''). DP clones below the PE are visible in these images. (C-C'') However, Myc-Lines was enriched in nuclei in the DP. (D,D') Flag-Bowl accumulated only in clones generated in the PE (arrow in D') and the ventro-anterior hinge (arrowhead in D'). Flag-Bowl failed to accumulate in clones generated in the DP. Bowl was stabilized in lines mutant cell clones (E-E'') and in drm expressing clones (F-F'') in the DP, and lost from the lines-expressing cell clones generated in the lateral margins of the PE (G-G'', arrow in G) and in the squamous PE (not shown). Clones were induced at 24-48 h AEL in A-D and at 72-96 h AEL in E-G.

View larger version (98K): [in this window] [in a new window]   Fig. 3. Overexpression of lines in the PE replaces the PE with DP. (A,C,D) Wild type; (B,E-G) Ubx-GAL4 UAS-Lines (Ubx>Lines); arrowheads in A,B,D,E indicate plane of z-section in A', B', D'-D''' and E'-E''', respectively. (A-B') Armadillo (Arm); cellular outlines are detected in apical sections; bright dots reveal adherence junctions in z-sections. Images in A and B are at the same magnification. (A-A') Wild type; (B-B') Ubx>Lines; the PE was replaced with a mirror image duplication of the pseudostratified CE and the disc was severely reduced in size. (C-E''') Dapi (blue); Nub (red) is restricted to the DP. (C,D) Wild type; optical sections at the level of the PE (C), and the DP (D). (D'-D''') The DP (left layer) expresses Nub and apposes the PE (right layer). (E-E''') Ubx>Lines; the PE (right layer) was replaced with a mirror-image duplication of the DP. Nub was detected symmetrically in both layers of these discs. (F) Ice (red) accumulated in clusters of apoptotic cells (arrows in F). Apoptosis is minimal in wild-type discs (data not shown). (G) Phospho-Histone H3 (PH3; red) stain reveals homogenous distribution of actively dividing cells in experimental discs.

View larger version (93K): [in this window] [in a new window]   Fig. 4. The DV, AP and PD patterning systems are elaborated in the absence of the PE. Analysis of Ubx>Lines discs with molecular markers for the PE and for the AP, DV and PD patterning systems. (A,E,G,I,K) Wild type; (B-D,F,H,J,L) Ubx>Lines; maximal intensity projections. Arrowheads in D,H,J,L indicate the plane of z-sections. (A) Wild type; Ubx (red) and Bowl (green) are expressed in the PE in nested domains. Ubx occupies a broad central domain. Bowl occupies a broader domain. (B-D) Ubx>Lines; expression of the peripodial markers Bowl (B) and Ubx (C) is absent; arrow in B indicates a leg where Bowl accumulation is not affected. (D) Lines was broadly nuclear. (E) Wild type; Vg (red) marks the pouch; Zfh2 (green) marks the hinge. (F) Ubx>Lines; Vg and Zfh2 were expressed in adjacent domains in both layers of the wing disc. The Zfh2-negative notum formed a tiny rudiment (arrow). (G) Wild type; Wg is restricted to the DP. (H) Ubx>Lines; Wg (red) was detected in the pouch (short arrow) and hinge (long arrows) in both layers of the disc epithelium but not in the notum. In wild type, (I) Dll and (K) Sal are induced in response to Wg and Dpp signaling, respectively. In Ubx>Lines, (J) Dll (red), Zfh2 (green) and (L) Sal were induced in both layers of these discs. Scale bar: 100 µm in B-D,F-L; 200 µm in A,E-K.

View larger version (118K): [in this window] [in a new window]   Fig. 5. Ectopic lines expression can completely transform the PE into DP, induce formation of secondary wings, and disrupt notal growth. (A,B) Wild type. (C-H) lines expressing clones (green) induced at the early first instar. Dapi (blue), Tsh (yellow), Wg, Nub or Al (red). (A) Tsh is excluded and Nub is restricted to the pouch and distal hinge. (B) Wg demarcates the pouch, the proximal and the distal hinge, and the medial region of the notum. (C) Large patches of lines-expressing cells in the DP had no effect on wing development. (D) Large patches of lines-expressing clones in the PE completely transformed the PE into DP. Arrowheads indicate the z-section shown in insets. Tsh and Nub were symmetrically expressed in both layers of the disc. (E,F) Smaller clones led to formation of secondary wings (arrows in right panels indicate secondary wings, arrowheads indicate Wg expression along the wing margin). These clones localized to the central region of the secondary wings. Large arrowheads indicate plane of z-section shown in insets. (F) In a subset of discs, the notum was reduced or absent. Asterisk indicates the loss of Wg expression in the presumptive notum. (G) Ptc-GAL4 UAS-GFP; GFP expression was detected along the AP compartment boundary in the DP and in the PE (marked by apposing arrows); Al marks the anterior part of the pouch. (H) Ptc-GAL4 UAS-GFP UAS-Lines; the Ptc-expressing cells in the PE adopted DP fate and permitted formation of secondary wings. Al and GFP were co-expressed in a subset of pouch cells.

View larger version (80K): [in this window] [in a new window]   Fig. 6. lines and bowl promote cell survival in the DP and the PE, respectively. (A,D) Control clones, (B,C) lines mutant FLP/FRT clones, (E) drm expressing clones and (F) bowl mutant MARCM clones. Dapi, blue in A-D,F. (A) Control and wild-type twin clones induced at the first instar survived in both the PE and DP. Optical section is at the level of the PE but DP clones are also visible. (B,C) lines mutant clones and twin spots survived in the PE (B), but mostly twin spots survived in the DP (C). (D) Control clones survived in both the DP and the PE. (E) drm-expressing clones were recovered in the PE but not in the DP. Wg expression (red) in the DP is visible. (F) bowl mutant clones survived in the DP but poorly in the PE. Arrowhead indicates a bowl mutant clone near the disc stalk. (G) Quantitative analysis of the recovery of wild-type, bowl and lines mutant clones. To calculate the the percentage recovery, we pooled the number of clones and separately the corresponding wild-type twin spots from several discs. L1, first instar; L2, second instar.

View larger version (79K): [in this window] [in a new window]   Fig. 7. Loss of lines function in the DP results in transformation of DP into PE fate. lines mutant clones generated at the second instar and stained for peripodial, hinge and pouch markers. (B,D-F) FLP/FRT and (A,C) MARCM clones; arrows point to magnified regions shown in right insets. Arrowheads in C'-C'' and D'-D'' indicate planes of z-sections shown in C'''-C'''' and D''', respectively. Images in A-B,E-F are projections. lines mutant clones occasionally overproliferated and protruded from the disc epithelium. (A,B) DP markers Nub (A) and Vg (B) were lost in these clones. The clone marked with an arrowhead in A,A' is below the Nub domain and lost Nub expression. (C-D''') Reciprocally, the PE markers Ubx (C) and Eya (D) were ectopically expressed in these clones. Ubx was detected only in posterior DP clones. (C''',C'''',D''') z-sections reveal basal extrusion of clones from the DP. (E) Tsh and (F) Hth, which mark the PE, the notum and the hinge, were ectopically expressed in these clones.

View larger version (130K): [in this window] [in a new window]   Fig. 8. lines specifies distal pouch fates and inhibits proximal hinge fate at the third instar. (A,C-H) Late third instar lines mutant FLP/FRT clones. (B) drm-expressing clones. Arrowheads in A and B indicate planes of z-sections shown in A',A'',B',B''. Arrows in C-H indicate magnified regions shown in insets. (A) Bowl (red) and GFP (green). (A-A'') lines mutant clones and (B-B'') drm-expressing clones extruded basally (arrows in A' and B'). (C-C'') Tsh, (D-D'') Hth and (E-E'') Wg, the expression of which overlaps in the hinge, were ectopically expressed in these clones. The expression of these proteins increased at a distance from the AP compartment boundary. (F-F'') Expression of Nub, which localizes to both the pouch and distal hinge was unaffected. (G-H'') The expression of the wing specific proteins (G) Dll and (H) Sal was lost from the clones.

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