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First published online 27 February 2007
doi: 10.1242/jcs.03404

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Integrin-ECM interactions regulate the changes in cell shape driving the morphogenesis of the Drosophila wing epithelium

¹ Institut de Reserca Biomédica, Barcelona 08028, Spain
² The Gurdon Institute and Dept. of PDN, University of Cambridge, Cambridge, CB21QR, UK
³ Centro Andaluz de Biología del Desarrollo (CSIC), Universidad Pablo de Olavide, Sevilla 41013, Spain

View larger version (55K): [in this window] [in a new window]   Fig. 1. Prepupal wing disc development. (A) Confocal section of a third-instar wing disc stained for filamentous actin with Rhodamine-phalloidin showing the apical region of the wing disc epithelium. The white dotted line outlines the central fold of the disc, the wing pouch, which will generate the wing blade. (B-D) Longitudinal sections of discs at 0, 2 and 4 hour after puparium (AP) formation that approximately correspond to the vertical white line in A. Arrowheads point to the wing pouch area. (B) At 0 hours, AP formation the wing disc is made up of a columnar epithelium (ce) that is continuous with the squamous epithelium of the peripodial membrane (pm). At this stage, a thin basal lamina (bl) surrounds the basal surface of disc cells (inset in B). (C) Folding of the wing pouch starts as cells in the wing margin (wm) shorten and separate from the basal lamina (inset in C). (D) Unfolding of the periphery of the wing pouch brings dorsal (d) and ventral (v) surfaces together, and simultaneously changes in cell shape from a columnar to a cuboidal morphology produce an increase in surface area.

View larger version (37K): [in this window] [in a new window]   Fig. 2. The expression of di in wing discs causes a dominant-negative effect and produces wing blisters. (A) Adults hemizygous for a viable hypomorphic mys allele, mysB43 do not develop wing blisters. (B) Expression of di in the whole wing disc using the CY2 GAL4 line causes a wing blister phenotype to develop in the adult. (C) Wing blistering induced by di overexpression is enhanced in an integrin mutant background, mysB43 (compare bubble in C with that in B). This enhancement is quantified in D.

View larger version (43K): [in this window] [in a new window]   Fig. 3. Expression of the chimeric di integrin in the wing imaginal disc induces cell shape changes, detachment of the basal lamina and disorganisation of the basal matrix. Wild-type wing discs (A-C) or wing discs expressing the di chimera in the posterior region of the wing disc (p) driven by the engrailed–GAL4 line (enG4: F,G,H,E,J). (D,I) di expressing clones in the wing pouch. (A,F) Filamentous actin staining with Rhodamine-phalloidin in early third-instar larval discs shows that expression of the di chimera leads to the formation of extra folds – compare the posterior side (p) with the anterior wild type (a). This was better visualised by scanning electron microscopy (B,G). (C,H) In cross-sections of wing discs, the expression of di clearly leads to a reduction in cell height (arrows in C and H point to the width of the epithelium). (D,I) Cells expressing di (green in D) also expand their apical cell surface as seen with an anti-phosphotyrosine antibody (red). (E) Localisation of laminin A to the basal side of wild-type cells is also affected by di expression, compare posterior experimental side (p) with that of anterior wild type (a) in either an xy (E) or an xz confocal (E') section. (J) Similarly, di expression affects the organisation of a second form of basal matrix. Bars, 8 µm (D,I) and in 30 µm (E,J).

View larger version (173K): [in this window] [in a new window]   Fig. 4. Expression of di inhibits adhesion between the two surfaces of the wing disc. (A,B) Longitudinal sections (1 µm) through prepupal wings at 4 hours after puparium formation. (A,A') The wild-type wing disc has already folded and the basal surfaces of the epithelial cells contact cells on the opposite surface via basal projections, except for a small gap at the marginal vein (*). (B,B') In wing discs expressing di in the whole wing pouch, the cells lack projections and do not come in contact with cells at the opposite surface. In addition, folding does not take place along the wing margin (wm) leading to a mismatch between dorsal (d) and ventral (v) surfaces (arrows). (C,D) Scanning microscopy of late third-instar wing discs reveals that wing margin cells in discs expressing di cannot be morphologically distinguished from the rest.

View larger version (73K): [in this window] [in a new window]   Fig. 5. Lack of integrin function in the wing disc reproduces the phenotype resulting from di overexpression. (A-F) Confocal sections of late third-instar wing discs containing groups of cells in the posterior (p) compartment lacking the PS subunit, marked by the absence of GFP (white in A and C) or PS (green in E). (B) Staining of filamentous actin with Rhodamine-phalloidin (RP) shows that lack of integrin function in the discs leads to the formation of extra folds. (D) A confocal section of a wing disc stained with an antibody that labels the basal matrix (ecm) shows that the matrix associated with the posterior PS mutant cells looks disorganised and hollow. (E,F) xz confocal sections of wing discs stained with Rhodamine-phalloidin (red) and an anti-PS antibody (green) shows that wing epithelial cells lacking the PS integrins (between arrows) are reduced in height when compared with the wild-type neighbours. Expression of PS in cells of the peripodial membrane (at the top) is not affected. (G) Cross-sections of these mutant discs show that the lack of PS induces a change in cell shape from a columnar to a cuboidal morphology (arrows indicate the height of the cells). Bar in A, 30 µm for A-D; a, anterior compartment.

View larger version (61K): [in this window] [in a new window]   Fig. 6. Expression of the metalloproteinase Mmp2 in the whole wing disc reproduces the phenotype resulting from a lack of integrin function. Staining of late third-instar wing discs with Rhodamine-phalloidin (A,B,A',B') show that ectopic expression of Mmp2 in the whole wing pouch (B,B') leads to the formation of extra folds. This can be clearly seen in xz confocal sections (A',B'). The white line in A and B indicates the position of the z section. (C) Scanning microscopy show that in some cases these extra folds seem to extend and form a pouch. (D,E) Confocal sections through the basal side of the wing pouch area of discs stained with an anti-PS antibody (green) show that the basal distribution of the endogenous PS integrins found in wild-type discs (D) is not affected upon Mmp2 overexpression (E). (F) Histological cross-sections show that expression of Mmp2 leads to a reduction in cell height (arrow indicates the height of the wing epithelium), resulting in wing blistering in the adult (G).

View larger version (105K): [in this window] [in a new window]   Fig. 7. Expression of di inhibits endogenous integrin localisation while recruiting talin to the basal membrane. (A,B) Late third-instar wing discs expressing di (green in inset in A) in a narrow band of cells along the dorsoventral axis of the wing disc using the patched-GAL4 line (ptcG4). (A) Confocal sections through the basal side of wing discs stained with an anti-PS antibody show that the expression of di affects the basal localisation of the endogenous integrins. (B) However, z sections show that the PS protein is still present on the lateral and apical surfaces. The white line in A indicates the position of the z section. (C-E) Confocal sections of late third instar wing imaginal discs expressing di in the posterior (p) compartment, stained for the PS subunit (green) and talin (red). In the anterior (a) wild-type half, talin colocalises with the PS subunit in basal focal adhesion-like structures (C). In the posterior compartment, di inhibits PS clustering (D), but not talin recruitment (E). In fact, di recruits talin more efficiently than the endogenous integrins (E, compare levels of talin in p with those in a). Bars, 30 µm (A), 20 µm (C-D).

View larger version (38K): [in this window] [in a new window]   Fig. 8. RafDN suppressed the effects of di on adhesion and cell shape. (A) Wild-type adult wing. (B) Expression of di in the whole wing pouch results in wing blistering. (C) Coexpression of RafDN rescues the wing-blister phenotype produced by di. (D-F) Longitudinal sections (inset in D) through third-instar wing discs. (D) Wild type. (E,F) Coexpression of RafDN (F) suppresses the change in cell shape produced by di (E). Arrows indicate height of wing disc cells. (G) Barr diagramme showing the percentage of rescue of the di phenotype by RafDN using different GAL4 lines.

View larger version (60K): [in this window] [in a new window]   Fig. 9. RafDN suppressed the effects of di on receptor localisation and matrix integrity. (A-D) Confocal sections of wing imaginal discs expressing di (A,C) or di+RafDN (B,D) using the patch-GAL4 line (ptcG4) (inset in A). (A) Staining for PS (green) shows that expression of di inhibits integrin recruitment to focal adhesions. (B) Coexpression of RafDN in these cells substantially rescues endogenous integrin distribution. (C,D) Coexpression of RafDN also suppresses the ability of di to disorganise the basal matrix (green), compare the appearance of the matrix within the patch domain in C with that in D. Bar, 20 µm. (E) In wild-type larval third-instar discs MAPK activation is detected specifically in all veins and in the wing margin. (F) In wing imaginal discs expressing di using the engrailed-GAL4 line (enG4) MAPK activity is now detected in the whole posterior compartment.