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The role of RBF in developmentally regulated cell proliferation in the eye disc and in Cyclin D/Cdk4 induced cellular growth

Shijie Xin1,*, Li Weng1,2,*, Jinhua Xu1 and Wei Du1,2,{dagger}

1 Ben May Institute for Cancer Research and Center for Molecular Oncology, The University of Chicago, 924 E. 57th Street, Chicago, IL 60637, USA
2 Committee on Cancer Biology, The University of Chicago, 924 E 57th Street, Chicago, IL 60637, USA
* These authors contributed equally to this work



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  		Fig. 1. (A) The putative cdk phosphorylation sites in RBF and the different RBF mutants generated. There are two other potential phosphorylation sites (T83 at the N terminus and T610 in the middle of the pocket domain) in RBF that are not mutated and are not shown. (B) The effects of Cyclin D/Cdk4 and Cyclin E/Cdc2c on wild-type RBF and RBF phosphorylation site mutants. SL2 cells were transfected with the E2F4CAT reporter construct, together with the E2F1/DP, Cyclin D/Cdk4, Cyclin E/Cdc2c, and the different RBF constructs as indicated. Basal indicates cells transfected with E2F4CAT reporter only. D/Cdk4 represents Drosophila Cyclin D and Cdk4. E/Cdc2c represents Drosophila Cyclin E and Cdc2c. ‘+’ indicates that a given construct was co-transfected; ‘-’ indicates that the construct was not co-transfected. (C) A yeast two-hybrid interaction assay to test the interaction between several RBF mutants and dE2F1. Various patches of yeast contained the pPC86-dE2F1 plasmid and the plasmids as indicated: Control, pPC97; WT RBF, pPC97-RBF(WT); RBF-30, pPC97-RBF-30; RBF-596, pPC97-RBF-596W; RBF-555, pPC97-RBF-555L. Patches of cells growing on plates selective for the presence of both plasmids were tested for the ß-galactosidase activity.

 


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  		Fig. 2. Scanning electron micrographs of the phenotypes of expressing different mutant forms of RBF. (A) Wild type. (B-H) Different RBF mutant constructs expressed using the eyeless GAL4 driver. (B) Wild type RBF; (C) RBF-223; (D) RBF-10; (E), RBF-30; (F) RBF-555L; (G) RBF-596W; (H) RBF-16. (I,J) GMRRBF-2804 at low (I) and high (J) magnification. (K) Wild type at high magnification. (L) GMRCyclin D/+. Arrowhead in J shows fused ommatidia; arrow shows an ommatidium with abnormal shape.

 


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  		Fig. 3. The effect of RBF-280 expression on E2F target gene expression and on mitosis. (A-D) In situ hybridization of wild-type and GMRRBF-2804 eye discs with antisense probes to PCNA allows for visualization of E2F target gene expression. (A,C) Low magnification showing whole eye discs. (B,D) High magnification view showing the details of the PCNA expression. (E-H) Anti-phospho-Histone H3 antibody staining of eye discs from wild-type and GMRRBF-2802 eye discs labels cells in mitosis. (E,G) Low magnification showing whole eye discs. (F,H) High magnification view showing the details of mitosis. In all the discs, posterior is oriented towards the right. Arrowheads indicate the morphogenetic furrow.

 


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  		Fig. 4. The effect of RBF-280 expression on S phase in the second mitotic wave. (A-D) Low-magnification view; (E-H) High-magnification view. (A,E) A wild-type eye disc; (B,F) A GMRRBF-280/GMRRBF-280 eye disc; (C,G) A GMRRBF-280/GMRDap eye disc; (D,H) A GMRDap/+ eye disc. The BrdU-positive cells in the second mitotic wave in wild-type eye discs are clustered together, indicating that cells finish S phase rapidly and synchronously. In GMRRBF-2804 eye discs, the second mitotic wave is broader and the BrdU staining is weaker in the second mitotic wave, indicating a prolongation of S phase and a loss of synchrony in the completion of S phase. (A,B,E,F) 1 hour of BrdU incorporation was used instead of 30 minutes to visualize the weaker staining in the second mitotic wave of GMRRBF-2804. In all the discs, posterior is oriented towards the right. Arrows indicate the second mitotic wave and arrowheads indicate the morphogenetic furrow.

 


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  		Fig. 5. Scanning electron micrographs (SEM) showing the adult eye phenotypes of GMRRBF-2802 and its interactions with Cyclin D and Cyclin E. (A) GMRRBF4C; (B) GMRRBF4C/GMRCyclin D; (C) dap/+;GMRRBF4C; (D) GMRRBF4C,GMRDap/+; (E) GMRGAL4/+;UASCyclin E/+; (F) GMRRBF-2802/+; (G) GMRRBF-2802/GMRCyclin D; (H) dap/+;GMRRBF-2802; (I) GMRRBF-2802,GMRDap; (J) GMRGAL4/+;UASCyclin E/GMRRBF-2802; (K,L) high magnification view of GMRRBF4C (K) and GMRRBF4C/GMRCyclin D (L); (M) GMRDap/+; (N) cyclin E/+; GMRRBF4C/+; (O) cyclin E/+; GMRRBF-2802/+.

 


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  		Fig. 6. RBF-280 inhibits CyclinD/Cdk4 induced ectopic S phase but not the large eye phenotypes in the Drosophila eye. BrdU staining (red) of eye discs with Cyclin D/Cdk4 overexpression (A-C), or RBF-280+Cyclin D/Cdk4 overexpression (D-F) showing RBF-280 blocks CyclinD/Cdk4 induced ectopic S phase. Clones of cells with CyclinD/Cdk4 or RBF-280+CyclinD/Cdk4 overexpression are marked by GFP (green). Arrowheads point to the second mitotic wave and arrows point to GFP marked clones. (G-K) Phenotypes of fly eyes with CyclinD/Cdk4 or RBF-280+CyclinD/Cdk4 overexpression as indicated.

 


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  		Fig. 7. RBF-280 inhibits CyclinD/Cdk4 induced growth and proliferation but not activated Ras-induced cellular growth in the proliferating wing disc cells. (A) Distribution of clone size and cell number/clone for the genotype given is shown. Cell doubling times (DT) are shown. Clones were analyzed 43 hours post-induction. Total number of clones analyzed (n): control, 48; Cyclin D/Cdk4, 51; RBF-280, 60; RBF-280+Cyclin D/Cdk4, 47. P*, comparison of clone area or cell number/clone between RBF-280+Cyclin D/Cdk4 and RBF-280. Clones with RBF-280+Cyclin D/Cdk4 expression occupy similar areas (P*=0.68) and have similar number of cells (P*=0.32) as the RBF-280 clones. Comparison of the clone area or cell number/clone between other genotypes, P<0.0001. (B) RBF-280 does not block RasV12-induced cellular growth. Clones were analyzed 43 hours post induction. Total number of clones analyzed (n): control, 52; RasV12, 47; RBF-280, 46; RBF-280+RasV12, 88. P#, comparison of clone area or cell number/clone between RBF-280+RasV12 and RBF-280; P##, comparison of cell number/clone between RasV12 and wild type. Comparison of the clone area or cell number/clone between other genotypes, P<=0.0001. (C) Examples of cell clones of each genotype are shown as indicated. Clones are marked by GFP (green). DAPI staining that labels the nuclei is shown in blue. Cell sizes were estimated by the average of clone area/cell number per clone for all genotypes. The ratio between cell sizes of indicated genotype over wild-type cell size is shown.

 




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