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First published online 5 November 2008
doi: 10.1242/dev.027789

Development 135, 3995-4001 (2008)
Published by The Company of Biologists 2008
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Notch signalling coordinates tissue growth and wing fate specification in Drosophila

Neus Rafel and Marco Milán*

ICREA and Institute for Research in Biomedicine (IRB), Parc Científic de Barcelona, Baldiri Reixac 10, 08028 Barcelona, Spain.


Figure 1
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  		Fig. 1. Failure to specify wing fate in the absence of Notch activity. (A,B,D-I) Late third instar wing discs (A,D,F,H) and resulting adult wings or nota (B,E,G,I) of the following genotypes: wild type (A,B), sd-Gal4; UAS-SerTM or sd-Gal4; UAS-DlTM (D,E), sd-Gal4; UAS-NdsRNA (F,G) and sd-Gal4; UAS-notum (H,I). Two wing discs per genotype are shown and labelled to visualize Nubbin (Nub, red) or Wingless (Wg, red), and Homothorax (Hth, blue) or Teashirt (Tsh, blue) protein expression. Wing territory (w), endogenous nota (nt) and duplicated nota territories (nt') are marked in A,D,F,H. Magnifications of the adult duplicated heminota (blue arrows) are shown in the right panels in E,G,I. (C) sd-Gal4; UAS-GFP early second instar wing disc. Note expression in the whole wing disc. (J) Histogram showing the percentage duplicated (Dp, blue) and non-duplicated (NDp, yellow) hemi-nota in different genotypes. Number of scored heminota: DlTM, n=398; SerTM, n=211; NdsRNA, n=69; notum, n=40; sgg, n=312. (K,M,N) sd-Gal4; UAS-SerTMGal80ts late third instar wing disc (M) and resulting duplicated adult nota (K,N) of larvae raised at 18°C and shifted to 29°C during the second instar (48-72 hours after egg-laying, AEL). Wing disc in M was labelled to visualize Nubbin (Nub, red) and Homothorax (Hth, blue) protein expression. (L) Histogram showing the percentage of duplicated (Dp, blue) and non-duplicated (NDp, yellow) hemi-nota in sd-Gal4; UAS-SerTMGal80ts adult flies raised at 18°C and shifted to 29°C during different developmental stages: first instar (L1, 24-48 hours AEL), first/second instar (L1/2, 36-60 hours AEL), second instar (L2, 48-72 hours AEL), second/third instar (L2/3, 60-84 hours AEL) and third instar (L3, 72-96 hours AEL). Number of scored heminota: 18°C, n=148; L1, n=118; L1/2, n=314; L2, n=230; L2/3, n=74; L3, n=142.

 

Figure 2
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  		Fig. 2. Notch acts through Wg to induce wing fate specification. (A-I) Late third instar wing discs and adult wings of the following genotypes: UAS-NdsRNA/+; ptc-Gal4·UAS-GFP/+ (A), ptc-Gal4·UAS-GFP/+; UAS-Axin/+ (B), UAS-NdsRNA/+; ap-Gal4·UAS-GFP/+ (C), ap-Gal4·UAS-GFP/+; UAS-Axin/+ (D), sd-Gal4; UAS-SerTM, UAS-Wg (E), sd-Gal4; UAS-SerTM, UAS-ArmS10 (F,G), sd-Gal; UAS-notum, UAS-NINTRA (H), sd-Gal4; UAS-sgg, UAS-NINTRA (I), and labelled to visualize GFP (green, A-D), Homothorax (Hth, blue) and Nubbin (Nub, red) protein expression. The penetrance of the phenotype was 100% in E-I. Number of scored wing discs was: E, 29; F, 17; H, 9; I, 13. Wing territory (w), endogenous nota (nt) and duplicated nota territories (nt') are marked in E-I. (J,K) Wild-type (J) and sd-Gal4, UAS-SerTM or sd-Gal4, UAS-DlTM (K) early second instar wing discs labelled to visualize vn-lacZ and wg-lacZ expression. (L) Histogram showing the percentage of duplicated (Dp, blue) and non-duplicated (NDp, yellow) hemi-nota in sd-Gal4; UAS-SerTM adult flies in a +/+, wgr0727/+ or vn10567/+ mutant backgrounds. Number of scored heminota: +/+, 211; wgr0727/+, 108; vn10567/+, 108.

 

Figure 3
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  		Fig. 3. Growth is essential for wing fate specification. (A-H) Late third instar wing discs and adult wing or nota of the following genotypes: sd-Gal4, UAS-DlTM, UAS-CycE (A,B), sd-Gal4, UAS-SerTM, UAS-CycE (C,D), sd-Gal4, UAS-notum, UAS-CycE (E,F) and sd-Gal4, UAS-hippo (G,H). Wing discs were labelled to visualize Nubbin (Nub, red) and Homothorax (Hth, blue) protein expression. The penetrance of the phenotype was 100% in A-F. Number of scored wing discs ranged between 7 and 11. Wing territory (w), endogenous nota (nt) and duplicated nota territories (nt') are marked in A,C,E. Blue arrows point to duplicated nota in F-H. (I) Histogram showing the size (in arbitrary units) of wild type, sd-Gal4, UAS-SerTM and sd-Gal4, UAS-SerTM, UAS-CycE early second instar wing discs raised under the same conditions. The average wing disc sizes and standard deviations were 1±0.3 (wild type), 0.67±0.14 (sd>SerTM) and 1.13±0.36 (sd>SerTM, CycE). sd>SerTM wing discs were significantly smaller than wild-type discs (P<10-6) and expression of CycE was able to rescue wing disc size when compared with sd>SerTM wing discs (P<10-8). Number of scored discs: wild type, 22; sd>SerTM, 40; sd>SerTM, CycE, 21. (J) Histogram showing the percentage of duplicated (Dp, blue) and non-duplicated (NDp, yellow) hemi-nota in sd-Gal4; UAS-SerTM adult flies expressing UAS-GFP (n=236), UAS-CycE (n=36), UAS-Stg (n=136), UAS-CycD,UAS-Cdk4 (n=64), UAS-dMyc (n=94) or UAS-PTEN (n=40).

 

Figure 4
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  		Fig. 4. Coupling growth and wing fate specification. (A-C) Illustration of the subdivision of the wing imaginal disc into wing (red) and body wall (notum, green) primordia by the signalling molecules Wingless (Wg, orange) and Vein (Vn, green). Ventrally expressed Wg induces the expression of wing fate marker genes, whereas dorsally expressed Vn blocks this activation. Wg represses Vn expression restricting it to the dorsal region. (A) Vn is expressed by a subset of cells (green), but it diffuses and reaches every wing cell in the early wing primordium. In this situation, ventral cells (orange) are not able to respond to Wg and do not differentiate as wing tissue. (B,C) Growth of the tissue induced by the activity of Notch pulls the sources of Wg and Vn apart. Now, ventral cells (orange) do not sense sufficient levels of Vn, so they respond to Wg and differentiate as wing tissue (red nuclei). (D-G) Early (D,F), mid (G) and late (E) second instar wing discs labelled to visualize wg-lacZ (antibody to β-Gal, green) and Nubbin protein expression (Nub, red; D,E), or Wg (green) and Teashirt (Tsh, blue; F,G) protein expression.

 

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© The Company of Biologists Ltd 2008