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First published online 14 April 2009
doi: 10.1242/jcs.044271

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Dpp signaling promotes the cuboidal-to-columnar shape transition of Drosophila wing disc epithelia by regulating Rho1

Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstr. 108, 01307 Dresden, Germany

View larger version (83K): [in this window] [in a new window]   Fig. 1. Dpp signal transduction activity correlates with apical-basal cell length in wing discs. (A-E) Schemes of x-y (A,C) and cross section x-z (B,D,E) views of early-second instar (A,B) and late third instar (C-E) wing discs. The wing disc pouch is shaded in grey. Numbers in E refer to folds. Schemes are not to scale. (F-H) x-y (F) and x-z (G,H) views of wing discs from larvae 48±3 hours (F,G) and 60±3 hours (H) after egg lay, stained as indicated. brk-lacZ activity is detectable in most cells of wing discs at 48 hours, but restricted to peripheral cells at 60 hours after egg lay (arrowheads). (I,J) x-y (I) and x-z (J,J',J") views of late third instar wing discs stained as indicated. Dpp signal transduction activity correlates with apical-basal cell length (double-sided arrows). Rho1 protein is enriched at the apicolateral side of the highly elongated cells in the middle of the wing disc pouch. (K) Apical-basal cell length and pixel intensity of brk-lacZ as a function of the position along the anteroposterior axis for the image shown in J. (L,M) x-z views of late third instar wing discs stained as indicated. The Rho1 sensor (PKNG58AeGFP; tub-GAL4 tubP-gal80ts UAS-PKNG58AeGFP 24 hours after temperature shift to inducing conditions) and Sqh-GFP are enriched at the apicolateral side of the highly elongated cells in the middle of the wing disc pouch. Images in this figure are shown with the anterior to the left. In these and all subsequent x-z sections, apical of the columnar cells is to the top. Dotted lines indicate the position of x-z or x-y sections. Scale bars: 10 µm (F-H); 50 µm (I,L,M); 25 µm (J).

View larger version (100K): [in this window] [in a new window]   Fig. 2. Cells in tkva12 bsk- clones are apically constricted and short, and are extruded from the epithelium. (A-I) Clones of tkva12 bsk- cells, identified in A and G by the presence of CD8-GFP, were induced at the second instar larval stage and stained as indicated 24 hours (A-D) or 48-60 (E-I) hours later. (A) x-z view. tkva12 bsk- cells are shorter along the apical-basal axis than control cells (double-sided arrows). (B) Magnified view of the boxed area in the middle panel of A. Vertical dotted lines isolate the clone. (C,D) x-y sections of B. tkva12 bsk- cells are apically constricted (arrowheads) compared with control cells (arrows). (E) x-z view. tkva12 bsk- cells are part of a deep epithelial fold. (F) x-y-section of E. (G) x-z view. tkva12 bsk- cells have lost contact with the zonula adherens of neighboring control cells and form cyst-like structures. (H) x-y section. (I) x-z view. E-cadherin is greatly reduced in mutant cells. Arrows indicate tkva12 bsk- cells surrounded by Viking-GFP. (J,K) Clones of brkXH mad- cells, marked by the absence of GFP (green), induced at the second instar larval stage and stained 60 hours later. (J) x-y view. (K) x-z-section of J. brkXH mad- cells display a normal apical-basal length. Scale bars: 10 µm.

View larger version (62K): [in this window] [in a new window]   Fig. 3. A cell-autonomous role for Dpp signaling in maintaining a highly elongated cell shape. (A-D) x-z sections of wing discs coexpressing Dad and p35 in the dorsal compartment at the indicated time after temperature shift to inducing conditions and stained as indicated. (A-C) Cells of the dorsal pouch region become shorter over time compared with the ventral control cells (double-sided arrows). (B) Epithelium forms an apical invagination a few cell rows into the dorsal compartment (inset). Cells in the apical invagination are apically constricted (arrowhead) compared with cells located more dorsally (arrow). (C) A deep epithelial invagination (arrow) formed at the dorsal-ventral compartment boundary. (D) A basal indentation containing PSβ-integrin formed (arrows). (E,F) x-z sections of control wing disc (E) and wing disc coexpressing Dad and p35 under the control of nub-GAL4 in the wing disc pouch (F) stained as indicated. Cells coexpressing Dad and p35 are shorter along their apical-basal axis than pouch cells of the control (double-sided arrows on the left hand side). Prospective hinge cells, which do not display nub-GAL4 activity, serve as an internal control (double-sided arrows to the right). The pouch region in F is smaller compared to the pouch region in E. Epithelial fold shown on the left in F corresponds to a normal ventral hinge fold. Numbers in A-F denote folds in the wing disc (compare with Fig. 1E). x-z sections in A-F and Figs 4, 5, 6, 7 are taken approximately through the center of the wing disc perpendicular to the dorsal-ventral compartment boundary; dorsal is shown to the right. (G,H) x-y sections of the pouch region of control wing disc (G) and wing disc coexpressing Dad and p35 under control of nub-GAL4 (H) stained for E-cadherin. Cells coexpressing Dad and p35 display an increased apical circumference compared to controls. (I) Quantifications of x-z cross-section area and apical-basal cell length. Ratios between dorsal and ventral cells in control wing discs (UAS-dad UAS-p35) and ap-GAL4 tubP-gal80ts UAS-dad UAS-p35 wing discs 48 hours and 56 hours after temperature shift to inducing conditions are shown. Cells coexpressing Dad and p35 are significantly shorter and display a moderately reduced cross-section area compared with control cells. Means ± s.e.m. are shown [n=9 wing discs (control); n=7 (48h); n=14 (56h)]; *P<0.001. Scale bars: 20 µm (A-F); 5 µm (G,H).

View larger version (87K): [in this window] [in a new window]   Fig. 4. Dpp signaling is required for elevated levels of Rho1 sensor activity at the apicolateral side of cells. (A-C,F) x-z sections of wing discs coexpressing Dad and p35 in the dorsal compartment 56 hours after temperature shift to inducing conditions and stained as indicated. (A) Apicolaterally localized moesin is reduced in dorsal cells (arrow) compared with ventral cells (arrowhead) and is strongly reduced in the apical invagination (asterisk). (B) The apical microtubule network is highly reduced in dorsal cells (arrows) compared with ventral cells (arrowhead) and is undetectable in cells located deep in the apical invagination (asterisk). (C) Apicolaterally localized Rho1 is reduced in dorsal cells (arrow) compared with ventral control cells (arrowhead). (D,E) x-z sections of wing discs coexpressing p35, PKNG58AeGFP (Rho sensor) and lacZ (D) or p35, PKNG58AeGFP and Dad (E) in the dorsal compartment 56 hours after temperature shift to inducing conditions, and stained as indicated. Rho sensor activity is reduced on the apicolateral side of Dad-expressing cells (arrow) compared with control lacZ-expressing cells (arrowhead). (F) Apicolaterally localized P-MRLC is reduced in dorsal cells (arrow) compared to ventral cells (arrowhead). Dorsal compartment is shown to the right in A-F. Scale bars: 20 µm.

View larger version (72K): [in this window] [in a new window]   Fig. 5. Increased Dpp signaling results in elevated Rho1 sensor activity at the apicolateral side of cells and in precocious cell elongation. (A) x-z section of early third instar control wing disc expressing CD8-GFP under the control of ap-GAL4 in the dorsal compartment and stained as indicated. Dorsal and ventral cells have a similar apical-basal cell length (double-sided arrows). (B-D) x-z sections (B,D) or a projection of x-y sections (C) of early third instar wing discs coexpressing TkvQ-D and CD8-GFP under the control of ap-GAL4 stained as indicated. Cells within the presumed pouch region coexpressing TkvQ-D and CD8-GFP are pseudostratified and longer (double-sided arrows), are apically constricted and display increased Rho1 at their apicolateral sides (arrow) compared with control ventral cells (arrowhead). (E-G) x-z section of early third instar wing discs coexpressing lacZ and PKNG58AeGFP (Rho sensor) (E), TkvQ-D and PKNG58AeGFP (F) or TkvQ-D (G) under the control of ap-GAL4 in the dorsal compartment and stained as indicated. In G, Sqh-GFP is, in addition, expressed under its own promoter to visualize MRLC. TkvQ-D-expressing cells display increased Sqh-GFP abundance and Rho sensor activity (arrows) compared with control cells (arrowheads). (H) x-z section of a late third instar wing disc coexpressing TkvQ-D and CD8-GFP under the control of ap-GAL4 stained as indicated. Cells coexpressing TkvQ-D and CD8-GFP are moderately longer than control cells (double-sided arrows). (I) Ratio of apical-basal length between dorsal and ventral cells in control wing discs (ap-GAL4, UAS-CD8-GFP) and wing discs coexpressing CD8-GFP and TkvQ-D in the dorsal compartment (ap-GAL4 UAS-CD8-GFP UAS-TkvQ-D) of early third instar and late third instar larvae are shown. Means ± s.e.m. are indicated (n=11 wing discs (early, control); n=15 (early, TkvQ-D); n=14 (late, control); n=4 (late, TkvQ-D); **P<0.001. (J) Ratio of apical to lateral pixel intensities for F-actin, Rho1, PKNG58AeGFP and Sqh-GFP staining of ventral control cells and dorsal cells coexpressing CD8-GFP and TkvQ-D. Means ± s.e.m. are indicated [n=14 wing discs (F-actin); n=9 (Rho1); n=5 (PKNG58AeGFP, control); n=8 (PKNG58AeGFP, TkvQ-D); n=8 (Sqh-GFP)]; *P<0.05; **P<0.001. Scale bars: 20 µm (A,B,D-H); 10 µm (C).

View larger version (89K): [in this window] [in a new window]   Fig. 6. Rho1 and myosin II affect apical-basal cell length. (A-F) x-z sections of late third instar wing discs expressing Rho1N19 and p35 (A), double-stranded RNA targeting Rho1 (Rho1dsRNA) (B), Rho1V14 and p35 (C), RhoGEF2 (D), sqhdsRNA and p35 (E) or sqhE20E21 and p35 (F) in the dorsal compartment, 44 hours (A,B), 8 hours (C), 17 hours (D), 48 hours (E) or 56 hours (F) after temperature shift to inducing conditions and stained as indicated. (A,B) Cells expressing Rho1N19 or Rho1dsRNA are more highly elongated along the apical-basal axis compared with control ventral cells (double-sided arrows). (C,D) Cells expressing Rho1V14 or overexpressing RhoGEF2 are shorter than control cells (double-sided arrows) and tend to be packed more regularly. Cells at the interface between the Rho1V14 or RhoGEF2 overexpression domain and control cells are even shorter (asterisk) and form an epithelial invagination (arrow). Cells expressing sqhdsRNA (E) are more highly elongated and cells expressing sqhE20E21 (F) are shorter compared with control ventral cells (double-sided arrows). (G) The ratio of apical-basal length between dorsal and ventral cells of the experiments shown in A-F. Means ± s.e.m. are depicted [n=14 wing discs (control); n=14 (Rho1N19, p35); n=5 (Rho1dsRNA); n=11 (sqhdsRNA, p35); n=13 (Rho1V14, p35); n=10 (RhoGEF2); n=6 (sqhE20E21, p35)]; *P<0.001. Scale bars: 20 µm.

View larger version (95K): [in this window] [in a new window]   Fig. 7. Decreased Rho1 activity rescues the shortening of Dpp signaling compromised cells. (A-C) x-z sections of late third instar wing discs coexpressing Dad and p35 with Rho1N19 (A), sqhdsRNA (B) or MbsN300 (C) in the dorsal compartment 52 hours after temperature shift to inducing conditions and stained as indicated. The apical-basal length of dorsal cells and control ventral cells is comparable (double-sided arrows). (D) The ratio of apical-basal length between dorsal and ventral cells of the experiments shown in A-C. Means ± s.e.m. are depicted [n=14 wing discs (control); n=3 (mbsN300, p35); n=8 (GFP, dad, p35); n=8 (Rho1N19, dad, p35); n=6 (sqhdsRNA, dad, p35); n=12 (mbsN300, dad, p35)]; *P<0.001. Scale bars: 20 µm.

View larger version (56K): [in this window] [in a new window]   Fig. 8. Dpp signaling and Rho1 in epithelial cell shape. (A) Schematic representation of the consequences of loss of Dpp signaling in tkva12 bsk- clones (dark brown) on epithelial morphology. tkva12 bsk- mutant cells initially constrict apicolaterally and shorten along their apical-basal axis, resulting in the formation of an apical epithelial invagination. At the same time, the basal epithelial surface indentates, juxtaposing the basal sides of mutant cells and neighboring wild-type cells. Subsequently, mutant cells lose junctional contact to neighboring cells and extrude from the basal side of the epithelium. Extruded cells form long actin-rich protrusions and are lost from the epithelium. The epithelium attains its normal shape. (B) Schematic representation of the consequences of a compartment-wide reduction in Dpp signaling on epithelial morphology. Schemes represent ap-GAL4 tubP-gal80ts UAS-dad UAS-p35 wing discs 48 hours (left) and 56 hours (right) after temperature shift to inducing conditions. Cells with reduced Dpp signaling are shown in dark brown. (C) Scheme depicting the role of Dpp signaling in the compartmentalization of Rho1 and apical-basal cell elongation. Blue arrows indicate cortical tension. Interpretative view of the localization of Rho1 (light blue). (D) The proposed mechanism controlling apical-basal cell length in wing discs. Black arrows in A and B indicate indentations of the basal epithelial surface. x-z views are shown.

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