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First published online November 21, 2008
doi: 10.1242/10.1242/dev.025635

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Growth regulation by Dpp: an essential role for Brinker and a non-essential role for graded signaling levels

Institut für Molekularbiologie, Winterthurerstr. 190, CH-8057 Zürich, Switzerland.

View larger version (30K): [in this window] [in a new window]   Fig. 1. Quantitative comparison of the growth behavior of wild-type, tkvQ253D and brk- clones. (A,A',E) Wild-type (wt) Drosophila twin-spots. (B,B',F) tkvQ235D/wt twin-spots. (C,C',G) brkM68/wt twin-spots. (D,D',H) brkM68/tkvQ235D twin-spots. (A-D) Illustrations of the volumes of twin-spots. Disc margin and folds are indicated by cyan lines. Representative twin-spots for each genotype were chosen. Volumes were defined and measured using Imaris software. The image in B highlights the importance of using the volume to measure clone size. Although the apical area of the tkvQ235D clone is similar to that of the wild-type clone, its volume is 2.9-fold larger. Scale bars: 50 µm. (A'-D') The locations of the measured twin-spots are indicated; lateral (blue), medial (orange). (E-H) Volume ratio of medial and lateral twin-spots of each genotype. Error bars represent 95% confidence intervals (CIs). Number of twin-spots measured (n): (E) medial, n=14; lateral, n=11; (F) medial, n=14; lateral, n=13; (G) medial, n=11; lateral, n=14; (H) medial, n=13; lateral, n=13.

View larger version (44K): [in this window] [in a new window]   Fig. 2. Comparison of cell proliferation, disc size and Dpp signaling activity between wild-type discs and discs with altered brk levels or Dpp pathway activity. (A-D'') Drosophila third instar discs stained for BrdU (A-D). Dpp signaling activity assayed by (A'-D') pMad staining (Tanimoto et al., 2000) and (A''-D'') brk-lacZ staining. Note that in A'' and B'', the brkx47-lacZ reporter (Campbell and Tomlinson, 1999) was used, which recapitulates brk expression but does affect brk functionally. In C'' and D'', the brkXA-lacZ allele was used, which recapitulates brk expression and disrupts brk function. (A'''-D''') Illustrations of Dpp levels, Brk levels and cell proliferation in discs from wt (A), C765>dpp (B), brkXA (C) and dpp12/14;brkXA double-mutant (D) animals. (E-G) dpp12/14 (E, DAPI), esg>brk (F, GFP) and esg>brk,dpp (G, GFP) discs illustrating the similar size of discs of these three genotypes. Note that activation of Brk with esg-Gal4 in the wing disc precursors blocks wing disc specification and therefore we blocked the system until the first instar stage (80 hours AEL at 18°C) using the Gal80ts system. (H) Comparison of the sizes of wild-type (n=37), C765>dpp (n=15), brkXA (n=27) and dpp12/14;brkXA (n=27) discs along the AP axis. (I) Comparison of the sizes of wild-type (n=13), dpp12/14 (n=27), esg>brk (n=13) and esg>brk,dpp (n=25) discs along the AP axis. Error bars in H and I indicate 95% CIs. Scale bars: 50 µm.

View larger version (36K): [in this window] [in a new window]   Fig. 3. Uniform activation of the Dpp pathway leads to enhanced proliferation in the lateral areas and reduced proliferation in the medial area. (A) Definition of the medial and lateral areas. Note that the lateral region overlaps with the outer region of the pouch and the medial region with the patched (ptc) expression domain (see Fig. S1A,B in the supplementary material). (B,C) Wild-type (B) and C765>dpp (C) Drosophila larva wing discs stained for phosphorylated histone H3 (pH-H3). (D) Quantification of pH-H3-positive cells in wild-type and C765>dpp discs. The numbers of pH-H3-positive cells in rectangles of 50x75 µm in the medial or lateral areas were counted. Wild-type discs, n=22; C765>dpp discs, n=27. (E-G) Comparative analysis of the behavior of wild-type lacZ-expressing clones in the medial and lateral areas of C765>dpp discs and wild-type discs. In some cases, owing to their size, lateral clones extended the defined border. (E) A C765>dpp disc containing medial and lateral clones (green). Volumes were calculated and are illustrated using Imaris software. (F) Comparison of the volume of medial and lateral clones induced 48 hours AEL in wild-type and C765>dpp discs. Wt: medial, n=25; lateral, n=24. C765>dpp: medial, n=45; lateral, n=17. (G) Comparison of the volume of clones induced 72 hours AEL. Wt: medial, n=61; lateral, n=29. C765>dpp: medial, n=139; lateral, n=34. Error bars (D,F,G) represent 95% CIs. Scale bars: 50 µm.

View larger version (18K): [in this window] [in a new window]   Fig. 4. In the wing disc, two areas with distinct growth properties are defined by a Dpp-independent mechanism. (A) esg>GFP discs at the second (inset) and third instar stage. The activity of the esg-Gal4 driver is uniform, with the exception of the notum, which is taken into consideration in our studies. (B) Disc of an esg>dpp Drosophila larva. (C) Bar chart showing the size of discs exposed to either uniform Dpp signaling from late second instar onwards (C765>dpp), discs exposed to uniform Dpp signaling from the beginning of wing disc development (esg>dpp), or discs lacking the Dpp-Brk system (dpp12/14;brkXA). Wild-type discs span 321±15 µm along the AP axis, C765>dpp discs 436±35 µm, esg>dpp discs 438±63 µm and dpp12/14;brkXA discs 438±21 µm (± 95% CIs). Wild-type discs, n=37; C765>dpp discs, n=15; dpp12/14;brkXA discs, n=27; esg>dpp discs, n=9. Scale bars: 50 µm.

View larger version (59K): [in this window] [in a new window]   Fig. 5. Graded Dpp pathway activity levels in the medial area of the wing disc are not required to drive cell proliferation. (A-A'') A second instar wing disc from a Drosophila larva expressing tkvQ235D in the medial area (salE-Gal4 driver). Gal4 function was inhibited during early embryogenesis by the Gal80ts system. Twenty-four hours AEL, embryos were shifted for 48 hours from 18°C to the permissive temperature of 29°C. (B-B'') Third instar wing discs of larvae expressing tkvQ235D under the salE-Gal4 driver. Gal80ts inhibited tkvQ235D expression during embryogenesis; 24 hours AEL, larvae were shifted for 96 hours to 29°C. (C-C'') The pMad and brk-lacZ gradient in wild-type wing discs. Quantification of antibody staining within the area of the green rectangles, from anterior to posterior, is shown in C''. (D-D'') pMad and brk-lacZ staining of salE>tkvQ235D discs (processed as in B); quantification is shown in D''. Scale bars: 50 µm.

View larger version (44K): [in this window] [in a new window]   Fig. 6. Ectopic Dpp signaling in the lateral area causes reduced proliferation in the medial area. (A-A'') tkvQ235D expression in a lateral AyGal4:PR clone. Gal4:PR activity was induced by the addition of progesterone from 72 hours AEL for 48 hours. (B-B'') tkvQ235D was expressed under the en-Gal4 driver for 24 hours from 120 hours AEL (at 18°C); the Gal80ts system was used to temporally control Gal4 activity. (C-D'') tkvQ235D was expressed under the en-Gal4 driver for 48 hours from 120 hours AEL (at 18°C). (E) Quantification of the number of pH-H3-positive cells in a rectangle of 75x50 µm in the lateral-anterior, medial-anterior, medial-posterior and lateral-posterior areas of en>tkvQ235D discs as compared with the corresponding regions in wild-type Drosophila discs. Note that in wild-type discs, proliferation is uniform across the disc (see Fig. 3A) and therefore, for simplification, only the mean proliferation levels are shown here. Wild-type discs, n=22; en>tkvQ235D discs, n=32. Error bars display 95% CIs. A, anterior; P, posterior. Scale bars: 50 µm.

View larger version (8K): [in this window] [in a new window]   Fig. 7. Model of growth regulation in Drosophila wing discs by the Dpp-Brk system. (A) In wing discs without the Dpp-Brk system, growth is uneven. Lateral cells have a growth advantage over medial cells and overproliferate. By an unknown mechanism, lateral overproliferation inhibits growth in the medial area. (B) In wild-type wing discs, Dpp smooths out these local growth differences. High-level Dpp signaling prevents the expression of the growth inhibitor Brk in the medial area but not in the lateral area. High levels of Brk in the lateral area curb proliferation to levels similar to those in the medial area. A, anterior; P, posterior.

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