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First published online March 6, 2009
doi: 10.1242/10.1242/dev.021428

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Lines is required for normal operation of Wingless, Hedgehog and Notch pathways during wing development

¹ Centro de Biología Molecular `Severo Ochoa' (C.S.I.C.-UAM), Universidad Autónoma de Madrid, Cantoblanco, E-28049 Madrid, Spain.
² Department of Physiology, Development and Neuroscience, Anatomy Building, University of Cambridge, Downing Street, Cambridge CB2 3DY, UK.

View larger version (77K): [in this window] [in a new window]   Fig. 1. Lin/Drm/Bowl regulatory interaction in the wing imaginal disc. (A-D) Schematic representation of the epithelial layers of the wing imaginal disc: peripodial epithelium (containing the squamous cells and the marginal cells) (A), longitudinal section (B), disc proper (C) and cross-section (D), showing SC in red, MC in green and DP in yellow. (E-G) In situ hybridization in a wild-type disc showing lin (E), drm (F) and bowl (G) transcripts. (H-K) Lin protein is in the nucleus and cytoplasm in DP cells (H) but is cytoplasmic in MC (arrowheads in H,I) and SC cells (K). (L) Bowl protein is stabilized in MC cells but is not present in DP cells. (M) Bowl expression in the PE.

View larger version (92K): [in this window] [in a new window]   Fig. 2. Functional interaction between Lin, Drm and Bowl in wing development. (A,A') Bowl protein expression (green) in a wing disc containing a linG1 clone. (B,B') Subcellular localization of Lin protein in Drm GOF clones (red in B). In a higher magnification of B, Lin protein (green in B,B') is relocalized to the cytoplasm. (C,C') Bowl protein stabilization (green in C,C') in Drm GOF clones (red in C). (D-D'') ap-Gal4>UAS-linRNAi wing disc shows the absence of Lin protein (blue in D, grey in D') and the stabilization of Bowl (red in D, grey in D''). (E-E'') en-Gal4>UAS-bowlRNAi leg disc (labeled with GFP, green in E,E') shows absence of Bowl protein in the P compartment (red in E,E''). (F) A sd-Gal4>UAS-bowlRNAi wing with Bowl expression knocked down in the wing pouch. (G) The sd-Gal4>UAS-linRNAi wing shows a severe phenotype in terms of reduction of wing size. (H) This lin- phenotype is suppressed by co-expression of UAS-linRNAi and UAS-bowlRNAi using sd-Gal4. Scale bars: 500 µm.

View larger version (78K): [in this window] [in a new window]   Fig. 3. Repression of Wg responses; P/D transformation of linG1 clones in the wing disc. (A-A'') Nub repression (green in A,A'') in linG1clones induced in a wing disc (lack of red, arrowheads in A-A''). Inset in A shows Nub expression in the wild-type wing disc. (B) Proximal part of an adult wild-type wing. (C) Proximal part of a wing containing linG1 clones labeled by the cuticle marker forked. Arrowheads indicate ectopic macrocheatae. (D,D') Hth ectopic expression (red) in MARCM linG1 clones overexpressing ArmS10 (labeled with GFP, green). (E,E') Nub repression (green) in random clones co-expressing Drm and dTcf (red in E). (F,H,J) Wild-type expression of Zfh-2 (F), Tsh (H) and Hth (J) in the wing disc. (G,G',I,I',K,K') Ectopic activation of Zfh-2 (red in G,G'), Tsh (green in I,I') and Hth (red in K,K') in linG1 clones (labeled with GFP in G and K, and lack of red in I). All clones were induced between 48 and 72 hours AEL.

View larger version (121K): [in this window] [in a new window]   Fig. 4. Deregulation of Wg and Notch responses at the DV border of the wing disc. (A,C,E) Expression of Sens (A), Wg (C) and Ct (E) in a wild-type wing disc. (B,B') Sens repression (red) in a wing disc containing linG1 clones (lack of GFP and outlined). (D,D') Wg is ectopically activated (green) in linG1 clones (red) induced in the wing pouch. (F,F') Ct activation (green) in some linG1 clones in the wing pouch (red). Very large clones do not activate Ct probably because they were induced before the onset of the DV border. (G-J') Wing discs containing transient ectopic UAS-drm clones (red) produced by the tubGal80ts technique. After 7 hours at the restrictive temperature, Bowl protein (green) (G,G') is stabilized. After 18 hours, Sens (H,H') is autonomously repressed (green) and Wg (see brackets in I,I') and Ct (J,J') are activated. (K-K'') Ectopic clones co-expressing Lin RNAi and Bowl RNAi (green). The effects of knocking down lin by ectopic Lin RNAi are suppressed by co-expression of Bowl RNAi.

View larger version (103K): [in this window] [in a new window]   Fig. 5. Drm/Bowl induces the ectopic activation of Hh in the wing disc. (A-A'') Hh (red in A and grey in A') and Ptc (blue in A and grey in A'') expression in a wild-type wing disc. (B-B'') Ptc and Hh expressions in a ptc-Gal4> tubGal80ts; UAS-drm/UAS-GFP wing disc (after 29 hours of Drm induction). Note the ectopic expression of Hh (red in B, grey in B') and Ptc (blue in B, grey in B'') in the A compartment (arrowheads). (C-C'') Expression of hh-lacZ (red in C and grey in C') and Wg (green in C and grey in C'') in a ptcGal>tubGal80ts; UAS-drm/hh-lacZ wing disc (after 29 hours of Drm induction). Hh and Wg are activated within the Ptc domain (arrowheads). (D-D'') UAS-linRNAi clones (lack of green and marked with broken lines) induced using Act>CD2>Gal4; tubGal80ts system (after 29 hours of linRNAi induction). hh-lacZ (red in D and grey in D') is activated only in the clones touching the AP border (arrowheads), and the repression of Sens (blue in D and grey in D'') is present in all clones touching the DV border (asterisks).

View larger version (130K): [in this window] [in a new window]   Fig. 6. Bowl sequesters Gro from the N and Hh repressors complexes. Effects of expressing Bowl or Bowl variants on Notch (A-D'), Hh (E-F') and Wg (G-J') targets genes. (A-D') Ct expression (green) in wing discs containing clones (red) expressing ectopic Bowl (A,A'), Gro and Bowl (B,B'), Bowleh1- (C,C') or Bowleh1-VP16 (D,D'). Ectopic Ct expression is associated with ectopic Bowl (A,A') but not with ectopic Gro and Bowl (B,B'), Bowleh1- (C,C') or Bowleh1-VP16 (D,D'). (E-F') Hh expression (green) in ectopic Bowl clones and Bowleh1- clones (F,F'), all labeled in red and outlined. (G-J') Sens expression (green) in clones expressing ectopic Bowl (G,G'), Gro and Bowl (H,H'), Bowleh1- (I,I') or Bowleh1-VP16 clones (J,J'). All clones are labeled in red (arrowheads). Bowl represses Sens (G) in the presence (G-H') or absence (I) of the Gro-binding domain eh-1. Replacement of the eh-1 domain by VP16 converts Bowl to an activator of Sens close to the Wg source. The absence of Ct expression in a Bowleh1- clone (C,C') is probably due to the block of both N and Wg pathways and Wg responses in most of the Bowleh1- clones (I').

View larger version (102K): [in this window] [in a new window]   Fig. 7. H or Mtv partially suppress the effects of ectopic Drm in the wing. (A-A'') ptc-Gal4> UAS-drm/hh-lacZ wing disc showing a huge overgrowth and the induction of hh (red in A, grey in A') and Wg (green in A, grey in A'') in the AP compartment region where Drm is induced. (B,B') ptc-Gal4>UAS-H;UAS-drm/hh-lacZ wing disc. The co-expression of H and Drm largely normalizes the overgrowth and the expression of hh-lacZ (red in B and grey in B') and partially rescues at the DV border by the ectopic Wg (green in B) caused by ectopic Drm. (C,C') ptc-Gal4>UAS-H;UAS-drm. Note that Sens is still repressed (red in C, grey in C'). (D,D') ptc-Gal4/UAS-mtv;UAS-drm/hh-lacZ wing disc. Overgrowth and activation of hh-lacZ are largely normalized (red in D and grey in D') but the ectopic Wg expression (green in D) is not. (E,E') ptc-Gal4>UAS-mtv;UAS-drm/UAS-GFP wing disc (GFP labels the Ptc expression domain in E). Sens repression is not normalized (red in E, grey in E'). The Ptc expression domain is marked with a broken line in all panels.

View larger version (69K): [in this window] [in a new window]   Fig. 8. Bowl requirement in the peripodial cells of the wing disc. (A-B'') Wing discs containing MARCM bowl2 clones (green) in the PE (A) and in the DP (B). Note that Ubx (red in A,B and grey in A',B') and Hth (blue in A,B and grey in A'',B'') expression is not modified in bowl2 clones in either the PE (A) or in the DP (B). (C-C'') PE cells of a wild-type disc showing the expression of Ubx (red in C and grey in C') and Hth (green in C and grey in C''). (D-D'') ubx-Gal4>UAS-bowlRNAi wing discs showing the expression of Ubx (red in D and grey in D'), Hth (green in D and grey in D'') and Nub (blue in D). Both the size of the PE and the Ubx and Hth expression domains are reduced. The outlined area corresponds to the DP cells expressing Nub (blue in D). (E) Wild-type adult wing. (F,F') ubx-Gal4>UAS-bowlRNAi wings. (G) Wild-type pnr expression domain (GFP in green) in the notum region. (H) Wild-type adult notum. (I) pnr-Gal4>UAS-bowl RNAi notum. Note the cleft in the notum and the disorganization of micro and macrocheatae.

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