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The Drosophila melanogaster gene brain tumor negatively regulates cell growth and ribosomal RNA synthesis

Deborah J. Frank*, Bruce A. Edgar and Mark B. Roth{dagger}

Division of Basic Sciences and Molecular and Cellular Biology Program, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
* Present address: Biology Department, Washington University, 1 Brookings Drive, St Louis, MO 63130, USA



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  		Fig. 1. brat expression pattern in brain and imaginal discs. Brains and imaginal discs were dissected from third instar larvae and hybridized in situ using a brat antisense RNA probe. (A) Brain with the ventral nerve chord to the left and the brain hemispheres to the right. (B) Antenna (left) and eye (right) disc. Black arrow, morphogenetic furrow; white arrows, neuronal preclusters. (C) Wing disc. (D) Leg disc. No signal was detected when the sense probe was used (not shown). Scale bars: 50 µm in A; 100 µm in B-D.

 


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  		Fig. 2. Rescue of ncl-1 with a brat transgene. Worms mutant for ncl-1, those carrying a brat transgene, and wild-type worms were visualized using Nomarski optics. The left image shows neurons anterior to the posterior bulb of the pharynx in a ncl-1(e1942) worm. The middle image shows neurons from the same region in a ncl-1(e1942) worm carrying an array containing KS+ncl5'&3'/brat. The right image shows the same region from a wild-type worm. In each panel, two nuclei are indicated with white arrows. Note that large nucleoli are visible in nuclei in the left image only. Scale bar: 5 µm.

 


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  		Fig. 3. brat inhibits cell growth. (A) Part of a wing imaginal disc in which mitotic recombination was induced with a 2 hour heat shock at 72 hour AED to generate clones of cells that are brat11/brat11 (black areas) and GFP/GFP (bright green areas). Clones are surrounded by unrecombined brat11/GFP cells (light green areas). Scale bar: 10 µm. (B,C) Flow cytometric analysis of dissociated wing imaginal discs containing control clones (B) or brat11 clones (C). The x-axis shows forward light scatter, a relative measure of cell size, and the y-axis shows cell counts. Representative data from one experiment in which a 2 hour heat shock was applied at 72 hours AED is shown. (D) +/+ (control) or brat11/brat11 clone areas were compared with the corresponding GFP/GFP sister clones. A 45 minute heat shock was applied at 48 hours AED and clones were measured at 115 hours AED. Mean area ratio is plotted with standard error indicated by error bars. Data from two independent experiments was compiled. Genotypes are: hs-FLP122; Ub-GFP FRT40A/brat11 FRT40A (A, C and ‘brat11’ in D) and hs-FLP122; Ub-GFP FRT40A/FRT40A (B and ‘control’ in D).

 


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  		Fig. 4. Increased nucleolar size and rRNA level in brat/brat cells. (A) Mitotic recombination was induced with a 90 minute heat shock at 48 hours AED. At 115 hours AED, wing imaginal discs were fixed and stained with anti-Nop60B antibody. Data from two independent experiments are shown with the area of nucleoli in GFP/GFP cells set to 100 for simplicity. Mean nucleolar area is plotted with standard error indicated by error bars. The numbers of nucleoli analyzed are as follows: Experiment 1 GFP/GFP, 83; brat/brat, 47; Experiment 2 GFP/GFP, 74; brat/brat, 63. The genotype is hs-FLP122; Ub-GFP FRT40A/brat11 FRT40A. (B) Larvae from the cross, y; brat14/CyOY+ X y w; brat11/CyoY+ were separated based on mouth hook color at 24 hours AED. When they became wandering third instars, the genotypes were confirmed and wing discs and brains were removed and processed for RNA and DNA isolation using TRIzol Reagent (Gibco BRL). Isolated RNA (standardized to isolated DNA) was applied to nitrocellulose using a slot-blot apparatus and probed with radiolabeled D. melanogaster rDNA PCR product. Quantitation was performed using a BioRad Molecular Imager FX. rRNA level in control brat/+ is set to 100 for simplicity. Average rRNA level and standard error are indicated. Ten samples of each genotype were obtained on five subsequent days; each sample consisted of 9-16 brains or 14-24 wing discs.

 


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  		Fig. 5. Effect of brat overexpression in the eye and wing. The top left image shows a scanning electron micrograph of the head of a control fly. The top right image is the head of a fly in which brat is overexpressed in the eye. The bottom left image is of a wing from a fly carrying the dpp-Gal4 driver alone. The bottom right image is of a wing overexpressing brat under the control of dpp-Gal4, which expresses Gal4 between veins LIII and LIV (Staehling-Hampton and Hoffmann, 1994). Positions of veins are indicated in the bottom left image (I-V). Note the decrease in area between veins LIII and LIV as indicated by the bracket. All flies are female. Genotypes are w; P[w+; ey-Gal4]/+; +/TM6B (top left), w; P[w+; ey-Gal4]/+; P[w+; UAS-brat(5)]/+ (top right), w; P[w+;UAS-brat(1A)]/Sp; P[w+; dpp-Gal4]/+ (bottom left) and w; Sp/+; P[w+; dpp-Gal4]/+ (bottom right). Scale bars: 100 µm (top) and 250 µm (bottom).

 


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  		Fig. 6. brat overexpression increases cell size but inhibits clone growth. (A-D) Flow cytometric analysis of dissociated wing imaginal disc cells containing clones overexpressing the indicated genes. In all graphs, the x-axis shows forward light scatter, an indicator of relative cell size, and the y-axis shows cell counts. Overexpression of P35 regularly results in a small decrease in cell size relative to controls that is probably due to a deleterious effect on cell metabolism. (E) Control or UAS-brat clones were induced at 48 hours AED and analyzed 67 hours later. Clone areas were measured using Adobe Photoshop and the average pixel area is plotted with standard error. Eighty-eight control and 77 UAS-brat clones were measured. Genotypes are y w hs-FLP122; Sp/+; Act>CD2>Gal4 UAS-GFP/+ (A), y w hs-FLP122; Act>CD2>Gal4 UAS-GFP/UAS-brat(5) (B), y w hs-FLP122; Sp/UAS-P35; Act>CD2>Gal4 UAS-GFP/+ (C and ‘control’ in E), and y w hs-FLP122; +/UAS-P35; Act>CD2>Gal4 UAS-GFP/UAS-brat(5) (D and ‘UAS-brat’ in E).

 


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  		Fig. 7. brat overexpression inhibits cell division. Control, UAS-brat(1A) or UAS-brat(5) clones were induced at 72 hours AED and analyzed 43 hours later. The number of cells per clone was counted and plotted as a percentage of the total number of clones analyzed for each genotype. Doubling time (DT) is indicated next to each label. The statistical significances of the differences between experiments and control are: P=8x10–10 for UAS-brat(1A) and p=2x10–13 for UAS-brat(5). Number of clones analyzed was 76 for control, 104 for UAS-brat(1A) and 116 for UAS-brat(5). Genotypes are y w hs-FLP122; Sp/UAS-P35; Act>CD2>Gal4 UAS-GFP/+ (black bars), y w hs-FLP122; UAS-brat(1A)/UAS-P35; Act>CD2>Gal4 UAS-GFP/+ (white bars) and y w hs-FLP122; +/UAS-P35; Act>CD2>Gal4 UAS-GFP/UAS-brat(5) (striped bars). Similar results were obtained in two independent experiments.

 

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