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First published online 15 September 2004
doi: 10.1242/dev.01385

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The brinker gradient controls wing growth in Drosophila

Francisco A. Martín, Ainhoa Pérez-Garijo, Eduardo Moreno^* and Ginés Morata

Centro de Biología Molecular CSIC-UAM, Universidad Autónoma de Madrid, Madrid 28049, Spain

View larger version (63K): [in a new window]   Fig. 1. Wing size correlates with Dpp activity. (A-D) Wing discs containing different levels of Dpp activity in the wing pouch. All the discs are doubly stained for wg and GFP. All discs are presented at the same magnification, as indicated by the band of wg expression (red) in the thoracic region, which is not modified in the genotypes used. The wing pouch is labelled green using the UAS-GFP construct, which is not shown in the figure for simplification, with the nub-Gal4 driver. (A) The amount of Dpp signal is twice that in B, and results in a larger disc. (C) nub-Gal4>UAS-GFP disc contains a normal amount of Dpp. (D) The elevated levels of dad antagonise Dpp activity and produce a very small wing pouch. (E) Wing disc of the same genotype as in B, showing BrdU incorporation concentrated in the lateral region. (F-I) Adult wing size is dependent on the activity levels of the Dpp pathway: the greater the activity, the larger the wing.

View larger version (87K): [in a new window]   Fig. 2. Wing size correlates negatively with brk expression. All the discs are doubly stained for GFP and brk (anti-ßgal). The right panels show brk expression in red. For simplification, the labels present only a partial notation of the genotype (see Materials and methods). (A,B) A nub-Gal4>UAS-tkvQD UAS-GFP disc with a large wing pouch that has little or no brk activity. (C,D) nub-Gal4>UAS-GFP of normal size and normal brk activity. (E,F) The reduction of Dpp pathway activity in nub-Gal4>UAS-tkvDN UAS-GFP results in a small wing pouch associated with high brk levels. (G,H) Further diminution of Dpp pathway activity in nub-Gal4>UAS-dad UAS-GFP produces a smaller wing pouch associated with an expansion of brk expression that covers the whole of the wing pouch.

View larger version (91K): [in a new window]   Fig. 3. Extra growth produced by brk mutant clones. (A,B) brk– clones (A, yellow; B, forked36) in the anterior (A) and posterior (B) compartments. The clones do not produce pattern duplications but do produce additional tissue. (C) A disc doubly labelled arm-lacZ (green) and BrdU (red) with a brk– clone (arrow) showing greater BrdU incorporation than surrounding cells. (D,E) Disc of genotype nub-Gal4>UAS-dad stained for wg (red) containing two brk– clones (arrows) marked by the loss of GFP (green). The two clones appear in the wing pouch (which is delineated by the internal wg ring in E) and overgrow, even though they have originated in the wing pouch where there is virtually no Dpp activity.

View larger version (101K): [in a new window]   Fig. 4. Brk represses growth in a concentration-dependent manner. (A,B) Adult wings and thoraces of the same genotype C765-Gal4>UAS-brk grown at 17°C (A) and 25°C (B). The greater activity of Gal4 at 25°C (B) produces more Brk protein, which results in less growth than at 17°C (A). (C-E) Sets of mature C765-Gal4>UAS-brk UAS-GFP wing, haltere and leg discs dissected from larvae grown at 17°C (C), 25°C (D) and 29°C (E). The size of the discs inversely correlates with temperature.

View larger version (124K): [in a new window]   Fig. 5. Brk suppresses the excessive growth caused by the constitutive activity of the Dpp receptor Tkv in wings. (A) brk activity in the wing blocks growth, whereas constitutive activity of the Dpp pathway in the wing cells (B) causes excessive growth. (C) The presence of Brk in the wing suppresses the effect of TkvQD.

View larger version (140K): [in a new window]   Fig. 6. Brk induces low levels of apoptosis in the wing disc. (A) A wing disc of genotype nub-Gal4>UAS-brk UAS-GFP doubly stained for GFP (green) and caspase 3 activity (red). The green fluorescence marks where brk is expressed. (B) Low level of caspase 3 activity (red) in brk-expressing cells. (C,D) A disc of the same genotype but stained for GFP and TUNEL. Only a minority of brk-expressing cells undergo apoptosis (red in D). (E,F) Wing disc of genotype nub-Gal4>UAS-brk UAS-p35 UAS-GFP stained for GFP and TUNEL (red). The green fluorescence marks the cell containing brk and p35 activity. This disc (F) shows unusually high apoptotic levels, but was chosen to illustrate the effectiveness of p35 in suppressing cell death (absence of red staining in area corresponding to green staining in E).

View larger version (130K): [in a new window]   Fig. 7. Discontinuous levels of Brk give rise to JNK-mediated apoptosis. The activity of the JNK pathway is monitored by the expression of the puc-lacZ insert. (A,B) Control hh-Gal4>UAS-GFP disc showing normal JNK activity, which is restricted to the proximal thoracic region. pucZ expression is shown in red. (C,D) Disc of hh-Gal4>UAS-brk UAS-GFP. There is puc expression (red) at the border of the brk-expressing cells (green), along the AP compartment boundary (arrows). There appears to be puc expression at both sides of the border, suggesting a non-autonomous effect. (E,F) Clone of brk– cells labelled by the loss of GFP (green) showing puc activity at the border (arrows). (G,H) Caspase 3 activity (red, arrows) in the border of a brk– clone (shown by loss of green staining). There are also some scattered caspase-positive cells in the vicinity of other brk– clones.

View larger version (93K): [in a new window]   Fig. 8. Brk reduces the rates of cell proliferation. The upper panels show two discs of genotype nub-Gal4>UAS-brk UAS-GFP doubly stained for GFP and PH3 (left) and GFP and BrdU (right). In both cases, there is a marked reduction of both PH3 or BrdU (red) in the cells expressing brk. In the lower panels, two discs of genotype hh-Gal4>UAS-brk UAS-GFP show a similar result.

View larger version (105K): [in a new window]   Fig. 9. Brk downregulates bantam. (A,B) Wing disc with normal brk activity stained for PH3 and the bantam sensor: the brighter green colour corresponds to low bantam expression (see main text). The arrow indicates a characteristic zone of low bantam levels located in the brk domain. The distribution of the PH3 dots in the disc is uniform, indicating the uniform cell proliferation levels in the disc. (C,D) Disc of nub-Gal4>UAS-tkvQD genotype showing more PH3 staining in the lateral region, which is associated with partial loss of bantam. (E,F) A brk– clone showing greater bantam activity (arrows), as indicated by the reduction in the level of green staining.