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First published online 1 February 2006
doi: 10.1242/dev.02243

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Fat and Wingless signaling oppositely regulate epithelial cell-cell adhesion and distal wing development in Drosophila

Manish Jaiswal, Namita Agrawal^* and Pradip Sinha

Department of Biological Sciences and Bioengineering, Indian Institute of Technology, Kanpur 20 80 16, India

View larger version (113K): [in a new window]   Fig. 1. PD gradient in the levels of DE-Cad and cell shapes in wing imaginal disc epithelium. (A) Wing imaginal disc displaying Wg (blue) and Q-vg-lacZ (green). (B,C) Cartoons of optical section along the XY (B) or the XZ (C) plane reveal, respectively, cell shape and relative position of AJs (green in C) along the AP axis of the columnar pseudo-stratified epithelium. Nuclei are shown in magenta (C). (D-D'') In cells flanking the DV boundary (arrow), levels of both DE-Cad (D and red in D'') and Arm-GFP (D' and green in D'') are upregulated (merge, D''). (E) A high-resolution image of the AJs marked by DE-Cad-GFP (grey) in the XY optical plane reveals PD gradation in both DE-Cad levels and apical cell shapes; cells of the DV boundary are marked by Wg (blue). (F-F'') XZ section across the DV boundary of a wing disc epithelium reveals upregulated DE-Cad (F) and F-actin (rhodamine-phalloidin, F') in the AJs of cells, which flank the DV boundary (arrow). In the merge (F''), the DV boundary is marked by BE-vg-lacZ (blue); nuclei are marked by DAPI (magenta).

View larger version (168K): [in a new window]   Fig. 2. Wg signaling regulates DE-Cad levels and epithelial cell-cell adhesion in wing imaginal discs. AJs are visualized by DE-Cad immunolocalization (red or grey, A-E',I-I') and F-actin staining (red or grey, F-G'). GFP (green) marks the domains where transgenes are misexpressed. (A,A') Somatic clones expressing Dsh display upregulation of DE-Cad and reduction in apical cell circumferences in their AJs (arrowheads in A). Inset displays XZ section of a Dsh-expressing clone (green) to reveal cell-autonomous upregulation of DE-Cad in the AJs. (B-D') Somatic clones expressing Wg display both autonomous and non-cell autonomous (arrowheads in B,B') upregulation of DE-Cad. (C,C') Magnified image of the clone shown in the boxed areas (B,B'). Broken line indicates the border of the Wg-expressing clones. DE-Cad level within the clone (red broken line in C') is higher than in the neighboring cells. (D,D') A Wg-expressing somatic clone in the hinge region of wing imaginal disc also displays apical cell shape changes within and outside the Wg-secreting clones, besides epithelial misfolding. (E-G') Expression of GPI-DFz2 (en-Gal4 X UAS-GPI-DFz2) downregulates DE-Cad levels (E,E') and also F-actin (F-G') in the posterior (P, green) when compared with the anterior (A) wing compartment. (G,G') XZ optical section (along the yellow line in F) revealing downregulation of F-actin in posterior wing compartment; arrow indicates the AP boundary. (H-I') In wing imaginal disc of Nts mutant larva, grown at a restrictive temperature, expression of Wg at their DV boundary (arrowhead, H) is extinguished. These discs (I) also do not display characteristic PD gradient in the levels of DE-Cad (for wild-type pattern, see Fig. 1E). Higher magnification of the boxed region in I reveals nearly uniform apical cell shapes along the PD axis (I'). (J-M) Somatic clones overexpressing Dsh (J) or a non-degradable form of ß-catenin/Arm, ArmS10 (K) or DE-Cad (L) display smooth clone borders, while those of clones expressing wild-type form of ß-catenin/Arm, ArmS2 (M) are `wiggly'.

View larger version (144K): [in a new window]   Fig. 3. Wg signaling regulates expression of DE-Cad. (A-C') DE-Cad-lacZ reporter, detected by immunolocalization of ß-galactosidase (red or grey), is upregulated in cells flanking the DV boundary (A'), which receive high threshold of Wg. Somatic clones expressing ArmS10 (GFP, green, B) and Wg (GFP, green, C) show cell-autonomous (B') and non-cell-autonomous (C', arrows) activation of the DE-Cad-lacZ reporter, respectively.

View larger version (145K): [in a new window]   Fig. 4. Ft regulates cell-cell adhesion and DE-Cad expression. ft mutant clones (A-C) are marked by loss of GFP (green). Discs are stained for DE-Cad (red or grey in B-B'',D-E') or DE-Cad-lacZ (red or grey, C,C'). (A) ft422 mutant clones display altered cell-cell adhesion with characteristic smooth clone borders, unlike the `wiggly' borders of their wild-type (ft+/ft+) twins (arrowheads, brighter green). (B-B'') In ft422 clones, DE-Cad is upregulated. Part of this clone (box in B) is shown at a higher magnification (B'') to reveal higher levels of DE-Cad in the AJs of cells lacking Ft (ft-/ft-) when compared with those of the neighboring wild-type (ft+/ft-) cells; broken green line marks the clone border. (C,C') ftfd clones display upregulated expression of DE-Cad-lacZ. (D-E') Misexpression of Ft (enGal4/UAS-ft) in the posterior (P) wing compartment (GFP, green) downregulates DE-Cad when compared with that in the anterior (A) wing compartment. Boxed area in D is shown at higher magnification in E,E'. The PD gradient is lost in cell shape in the posterior wing compartment where Ft is overexpressed (see Fig. 1E); arrowheads mark the DV boundary.

View larger version (74K): [in a new window]   Fig. 5. Ft regulates PD wing growth and pattern. (A,B) Expression of Dll (red) and Wg (green) in wild-type (A) and in ftfd mutant (B) wing imaginal discs. Dll is upregulated in the ft mutant discs (B). (C,C') In the posterior (P) wing compartment displaying overexpression of Ft (UAS-ft/en-Gal4), Dll (red in C, grey in C') is downregulated when compared with its anterior (A) compartment or with that of a wild-type counterpart (also see Fig. 8H); the broken line (C') indicates the AP boundary. (D,D') A large ft422 clone, marked by loss of GFP (green), displays upregulated Q-vg-lacZ (red in D and grey in D'). (E) A plot of the intensity of Q-vg-lacZ expression over the wing pouch region (along the red broken line in D') to reveal upregulation of Q-vg-lacZ in the mutant clone along the PD axis. (F,G) Adult wing of a wild type (F) and of genotype en-Gal4/UAS-ft (G). Wing growth along the PD axis in the posterior compartment is truncated (G), as revealed by the close proximity of the anterior and posterior crossveins (arrowheads); AP boundary is indicated by broken lines.

View larger version (72K): [in a new window]   Fig. 6. ft genetically interacts with Wg signaling. (A) Expression of ArmS10 (UAS-ArmS10) under the regulation of the vg-Gal4 driver induces ectopic wing margin specific bristles in the wing blade. (B) This phenotype is enhanced by a reduction in the dose of ft (+/ftfd). (C,D) Expression of a dominant-negative form of dTCF (UAS-dTCFN) under the vg-Gal4 driver induces loss of wing margin bristles (C); this phenotype is partly suppressed by a reduction in ft gene dose (d). (E-H') ß-Catenin/Arm levels (red or grey) respond to the levels of Ft. ft422 mutant clones, marked by loss of GFP, display elevated levels of ß-catenin/Arm (E,E'). (F,F') Higher magnifications of some of these ft mutant clones (box in E) are displayed; insets show XZ projections through one such mutant clone to reveal upregulation of ß-catenin/Arm in ft mutant cells (non-GFP); broken line marks the clone border. Overexpression of Ft (en-Gal4/UAS-ft) (green, G-H') lowered the ß-catenin/Arm levels in the posterior (P) wing compartment compared with that of the anterior (A) wing compartment. (H,H') XZ section across the AP compartment of this disc (broken line in G,G') reveals downregulation of ß-catenin/Arm in the posterior (P) compartment.

View larger version (102K): [in a new window]   Fig. 7. Ft does not regulate Wg expression in the distal wing. (A) Expression of Wg (red) in the DV boundary (arrowhead) and in the inner (IR) and outer (OR) rings of the hinge region (arrows) in wild-type disc. (B) In ftfd mutant disc, levels of Wg expression in the DV boundary are comparable with that of wild-type discs (A), while in the hinge region (arrows) its expression is upregulated and broadened. (C-C') ft422 mutant clones (marked by loss of GFP, green) spanning the DV boundary (asterisks) do not affect Wg (red) expression, while in the hinge region its expression is upregulated (arrows).

View larger version (116K): [in a new window]   Fig. 8. Ft intersects Wg signaling downstream of the Wg ligand. (A-C',E). Expression of Wg (red) in the DV boundary (arrow, A), Q-vg-lacZ (green, A) and Dll (green, E) in the pouch region is extinguished in vg1 mutant wing discs. Misexpression of Wg (B) or loss of Ft in somatic clones (C) activates Q-vg-lacZ (B,C', green) in vg1 mutant wing discs. Higher magnification (C') of boxed area in C to reveal activation of Q-vg-lacZ (green) in ft mutant clones (ftfd/ftfd, marked by loss of GFP, blue). (D,F-G') ftfd vg1 (D,F) or Nts/Y; ftfd/ft422 (G,G') mutant wing imaginal discs do not express Wg (red) in their presumptive DV boundaries (arrows). Q-vg-lacZ (D',G'; green) and Dll (F, green) are activated in these discs. (H,H') Dll (red) and en-Gal4 (green) expression pattern in wild-type wing imaginal discs. Broken lines in this and subsequent figures indicate the AP compartment boundary. (I-J') Expression of GPI:DFz2 in the posterior wing compartment (en-Gal4/UAS-GPI:DFz2, green) downregulates the expression of Dll (red) in wild type (I,I') but not in the ftfd mutant disc (J,J'). (K,L) fzH51, fz2C1 double mutant clones induced 48-72 hours after egg laying in wild type (K) and ftfd mutant (L) wing imaginal discs do not survive, as revealed by the absence of non-GFP cells, whereas their twins do (cells with higher levels of GFP). (M-M') fzH51, fz2C1 double mutant clones in ftfd mutant discs can be recovered when induced 24 hours prior to dissection. These clones (arrows), however, do not show Dll expression (red).