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First published online 16 November 2005
doi: 10.1242/dev.02148

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Growth and cell survival are unevenly impaired in pixie mutant wing discs

¹ Cancer Research UK London Research Institute, PO Box 123, 44 Lincolns Inn Fields, London WC2A 3PX, UK
² National Centre for Biological Sciences, UAS-GKVK Campus, Bellary Road, Bangalore 560 065, India
³ Department of Genetics, Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC 27710, USA

View larger version (83K): [in a new window]   Fig. 1. pixie is an enhancer of a KD-Dp110-induced small-wing phenotype and shows Minute-like characteristics. (A-D) MS1096-Gal4 driven expression of UAS-KD-Dp110 reduces wing size and this phenotype is enhanced by pix3c2/+ and pix3c3/+. The genotype MS1096>KD-Dp110 is abbreviated to KD-Dp110. (E) Bar chart showing the average areas of the wings in A-D, n=8-12. (F,G) pix3c2/+ bristles (G) are shorter and more slender than control (F). (H-K) pixie mutant and M(3)66D/+ wings. Patterning defects are absent. Controls match the genetic background of the mutations, CS, CantonS. (L,M) Bar charts showing average ratio of wing/thorax circumference (L) and wing area (M) for the genotypes in H-K, n indicates numbers of flies/wings analyzed. When compared with control, the ratio in L is higher in pixL17/L35 (pix) flies and M66D1/+ (M) flies (P=3.4x10-10 and 0.0008, respectively). pixie mutant wing area is not significantly different from control, whereas M66D1/+ wings are significantly larger than control (P=0.15 and 3.7x10-25, respectively).

View larger version (26K): [in a new window]   Fig. 2. pixie encodes an ABC protein required for growth and translation. (A) A tree diagram illustrating the sequence similarity of the ABC domains of Pixie with those of other ABC-E and ABC-F proteins from Drosophila (Dm), Saccharomyces cerevisiae (Sc) and humans (Hs). (B) Predicted domain structure of Pixie, showing approximate positions of mis-sense mutations used in this study: pixL24 (Pro38Leu), pixL17 (Ala77Thr), pixL35 (Pro146Leu), pix3c2 (Gln231Leu) and pix3c3 (Gly316Asp). Pro38 (L24) and Ala77 (L17) are highly conserved consensus residues of the iron-sulphur binding domains. (C) Graphs show the impact of various dsRNAi treatments on global translation, measured as [35S]cysteine and methionine incorporation into total cellular protein, and represent combined data from three independent experiments. Relative translation/mg protein is expressed as a percentage of the control. In unpaired Student's t-tests, control and GFP do not differ; Pixie, eIF4A and emetine are significantly lower than control, *P<0.0001. Depletion of Pixie and the translation initiation factor eIF4A was confirmed by western blotting and immunofluorescence (see Fig. S1 in the supplementary material; data not shown). Depletion of eIF4A or Pixie reduced translation by day 2, before any effect on cell number (data not shown) or cell cycle profile. Lower panels show cell cycle profiles of pixie RNAi-treated (thick line) and GFP RNAi control (thin line) cells obtained through FACS analysis for one of the above experiments. By day three, pixie RNAi treatment increases the G1 population. (D) Western blots of various fractions of an 0.8 M sucrose cushion through which S2 cells lysates were spun. Lysate, nuclei-free lysate; top, fraction above the cushion; C, cushion; P100, pellet obtained in presence of 100 mM KCl; P500, pellet obtained in presence of 500 mM KCl. Akt is not detected in the ribosomal pellet, whereas Pixie is (but not in the presence of high salt levels).

View larger version (112K): [in a new window]   Fig. 3. Pixie is cytoplasmically localized and expressed uniformly in wing discs throughout development. Confocal images of wing discs immunostained with antiserum against Pixie. z sections are shown with x- and y-axis sections above and on the right respectively. The red line indicates the position of the y-section and the green line indicates the position of x-section. The blue line in the x- and y-sections depicts the position of the z-section. (A) Early third instar wing disc. (B) Wandering third instar disc. Additional panels above and on the right show in addition, propidium iodide staining (red), indicating the presence of nuclei in regions of the disc where Pixie staining is not detectable. Scale bar: 50 µm.

View larger version (121K): [in a new window]   Fig. 4. pixie mutant and Minute wing imaginal discs show regionally elevated levels of apoptosis. (A-L) Images are projections of confocal z-series taken through TUNEL-labelled wing imaginal discs that have been then inverted to reveal wing disc morphology, disc genotypes are indicated. Discs from (A,B) early to mid-third instar, (F,H) late third instar and (C-E,G,I-L) wandering larvae. Apoptosis is similar to control in discs heterozygous for pixL17 (D) and pixL35 (E), and is enhanced in discs that are transheterozygous for pixL17 and M(3)66D1 (J) and pixL17 and M(3)95A1 (L). Thin arrow in G indicates apoptotic nuclei that lie approximately at the DV boundary; thick arrow indicates those that lie at the pouch borders. (M) Bar charts and wing images showing that expressing p35 in the posterior compartment increases area in pixL17/L35 wings (pix) compared with control (pixL17/+). P/A ratio in pix wings is significantly higher than control, P=3.8x10-10. White line in images on the right indicates the AP boundary. (N) Wg protein, detected by immunostaining, is ectopically expressed (arrow) when p35 is expressed in the posterior compartment of pixie mutant discs. Scale bar: 50 µm in A-L.

View larger version (64K): [in a new window]   Fig. 5. pixL17 causes a reduction in balanced growth and cell survival in wing discs. (A) A pixL17, f36a wing clone (red) induced 27 hours before larval wandering and its accompanying mwh twin clone (blue). The presence of the mutant clone does not affect the arrangement of wing hairs in rows, indicating that mutant cell size is not altered. Bar chart on the right shows frequency and cell number of pixL17 wing clones (black bars) compared with their twins (grey bars). (B) GFP-, pixL17 clones and their GFP+/+ twins (examined at wandering stage and induced 46 hours before) in wing discs expressing the capsase inhibitor p35 in the posterior compartment (labelled using anti-En, red or orange when merged with GFP). (C) Apical (above) and basal (below) confocal sections through the same disc showing that nuclei of surviving pixL17 clones (white arrow) in the posterior pouch are more basal than their accompanying twins (black arrow). (D) Bar charts representing the number of cells in pixL17clones (black), and their twins (grey) in anterior clones (above) and in posterior p35-expressing clones (below); pouch clones are on left and hinge clones on right. TUNEL labelling confirmed that p35 expression blocked apoptosis (data not shown). Although posterior mutant clones are larger, the average mutant/twin clone size in the posterior compartment is still significantly less in the pouch (0.17) than the hinge (0.33), P<0.0001.

View larger version (45K): [in a new window]   Fig. 6. Spatial and temporal dynamics of the effect of pixL17 and eIF4A1006 on wing disc clonal growth. (A) A schematic showing the different clone induction windows used. Larval stage and developmental time in hours AEL is shown below. Black bars above indicate start and end of each clone induction window; duration is shown in hours (h) on right of each bar. Windows representing the `fast' phase end at mid-third instar (pink box); those representing the `slow' phase end at larval wandering and are within the blue box. The 60-hour window spans both phases. The boxes overlap; however, there is a time lag between the time of clone induction and clone generation, and MDT analysis (D) reveals that the actual overlap in the life of the clones is likely to be minimal (see Materials and methods). (B,C) Images of pixL17/GFP heterozygous discs with clones at mid-third instar (B) and a wandering third instar (C). The wing pouch, recognized as the unfolded epithelium at the centre of the disc, is marked by a white line; a blue line marks the DV boundary. The region regarded as hinge in our experiments surrounds the pouch and is within the red line. It includes regions that contribute to the ventral thorax, and, in early discs, the distal notum. (D-G) Bar charts in grey show the wild-type twin (GFP+/+) MDTs. Black and blue bar charts show mutant/twin clone size as average (black bars) and median (dark blue bars) with unfilled bars on right of each chart depicting a ratio of 1, which would indicate no effect on growth, for comparison. When clones are frequently absent, the median clone/twin size is 0, or much smaller than the average clone/twin size. As shown in A, pink and blue boxes mark clones examined at mid third instar or at wandering, respectively; the region examined (pouch or hinge) and genotype of clones (pixL17 and eIF4A1006) is indicated. x-axis shows clone induction window lengths (h), the median number of divisions within the twins (red) and the number of twins analyzed (n). Fig. S4 in the supplementary material shows raw clone and twin cell number data for the above experiments.

View larger version (33K): [in a new window]   Fig. 7. A Minute background has different effects on the growth of pixL17 clones during fast and slow growth. (A,B) Schematics showing the stages of development and clone induction windows used. Blue boxes represent discs examined at larval wandering and pink boxes discs examined at approximately mid-third instar. (C-L) Bar charts depicting clone size distributions of pixL17, Minute+ clones (C-J) and wild-type clones (K-L) as control. When Minute+/+ (Minute+) clones are generated in a Minute+/- (Minute) background, the Minute-/- twins survive poorly and cannot be used to assess background tissue growth. Thus, the size distribution of clones in a Minute background (right) is compared with the size of pixL17 or wild-type clones generated in a normal, Minute+ background (left), using comparable clone induction window lengths and stages of development. Minute-/- twins survive better during the fast phase and are shown as red bars in D. In the other experiments, these twins are rarely found. For example, among the clones depicted in H, four in the hinge were accompanied by twins. They are even more rare in the experiments depicted in F and L and are not seen in J. Twin clones in the Minute+ background are not depicted because this data is shown in Fig. 6. x-axes show size categories of clone cell number and y-axes show number of clones in each category. When sufficient numbers of clones are present, MDT is indicated. Difference in clone growth between pouch and hinge in D,F and L is significant, P<0.005 in D and F; P<0.000001 in L.

View larger version (22K): [in a new window]   Fig. 8. Model depicting changing levels of growth activators and inhibitors synthesized by disc cells in relation to increases in cell number. This model is a variation of a model proposed by Nijhout to explain how rising levels of growth inhibitors may determine disc size (Nijhout, 2003). In Nijhout's model, imaginal disc cells produce growth activators and inhibitors, and the inhibitors sequester or inactivate the activators. The level of each factor depends on the number of cells at the time and the influence of the inhibitor over the activator. At first activator levels are high, inhibitor levels low and disc growth is approximately exponential. When inhibitor levels become sufficiently high, growth begins to slow. Nijhout's model proposes that inhibitor activity remains high throughout slow growth. Accordingly, if pixie mutant cells were sensitive to the presence of inhibitor, then pixie mutant clone growth would be expected to worsen towards the end of larval life and during metamorphosis as growth slows. Instead, pixie mutant clone growth (survival) improves at the end of larval life (see text and Fig. 6E,F), and the intensity of cell death in pixie mutant and Minute discs decreases shortly before pupation (Fig. 4). Thus, we propose that the presence of activator is necessary for inhibitor synthesis. When inhibitor levels reach a threshold, this triggers the slowing down of growth and inactivation of the activator, which in turn leads to downregulation of inhibitor synthesis. Thus the pixie mutant clone phenotype is strong at the beginning of the slow phase, when inhibitor levels are high, but weak at the end of larval life when inhibitor levels are low, and growth is slow. The graphs presented here potentially apply to both the hinge and the pouch, with higher levels of inhibitors being found in the pouch than the hinge.

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