Figure 7. Elevated E2F activity in cells with inactivated Hippo pathway.

Clones of mutant cells were induced with ey-FLP. Position of the morphogenetic furrow (MF) is shown by arrowhead. Posterior is to the right. (A–C) Expression of the E2F reporter, PCNA-GFP, (red) in the wild type eye disc (A), or in the eye discs containing clones of wts mutant tissue (B) and de2f1 wts double mutant tissue (C). (A) In wild type disc, the E2F reporter is expressed in a narrow stripe (red) immediately posterior to the MF, preceding S phase entry into the second mitotic wave (SMW). (B) In wts mutant cells which are marked by the absence of β-Gal (green), the E2F reporter is inappropriately expressed in the posterior region of the eye disc. Mutant tissue is outlined. (C) Inappropriate expression of the E2F reporter in the posterior region of the eye disc is absent in de2f1 wts double mutant cells. Note, that clones of wts de2f1 double mutant cells were marked with β-Gal (green) produced from the de2f1⁷²⁹ mutant allele. de2f1 wts double mutant tissue is outlined. (D) Endogenous dE2F1 (magenta) is expressed within the MF in a wild type disc as revealed by anti-dE2F1 antibody. (E–G, I) Clones of mutant cells were induced with ey-FLP and mutant tissue is identified by the lack of GFP (green). (E) The anti-dE2F1 antibody is highly specific as the staining is absent in de2f1 mutant tissue (lack of green in E and pointed by the arrow). (F) wts and hpo mutant cells have an increased level of dE2F1 within the MF and inappropriately express dE2F1 posterior to the MF. Examples are pointed by the arrows. Position of mutant tissue is outlined. (G) Expression of endogenous dE2F1 protein (magenta) is unaffected in ago mutant cells in larval imaginal eye discs. (H) cyclin E was expressed ectopically in wild type mitotic clones using the MARCM system. Ectopic expression of cyclin E fails to elevate level of dE2F1 protein (magenta) posterior to the MF. Cells that express cyclin E are marked with GFP (green) and are outlined. (I) Endogenous dE2F2 protein (red) is expressed ubiquitously throughout the eye disc. Level of dE2F2 protein remains the same in both wts mutant and wild type tissue. (J) de2f1 is transcriptionally induced in hpo mutant cells as revealed by the de2f1 enhancer trap allele, de2f1⁷²⁹. de2f1⁷²⁹ contains the lacZ insertion into the endogenous de2f1 gene. The lacZ expression reflects transcription from the de2f1 promoter [34],[35]. Staining with anti-β-Gal antibody (magenta) was used to reveal expression of the lacZ gene in de2f1⁷²⁹. (K) SL2 cells were treated with nonspecific (NS), dE2F1 (E1), Warts (Wts) and Hippo (Hpo) dsRNA to deplete the corresponding proteins by RNAi. Cell extracts were analyzed by Western blot using antibody specific for Wts, dE2F1 and dE2F2. Depletion of Wts and Hpo shows an increase in the level of dE2F1 protein. In contrast, the dE2F2 protein level is not affected. The same blots were re-probed with anti-Tubulin antibody to control for equal loading. (L) Endogenous E2F activity is elevated in Hpo or Wts depleted SL2 cells. SL2 cells were incubated with non-specific (NS), RBF1, Hpo, and Wts dsRNAs for 4 days to deplete the corresponding proteins. On day 4, the E2F reporter (PCNA-luc) was transfected into the depleted cells and the luciferase activity was measured 2 days later to determine the level of the endogenous E2F activity in these cells. The pIE-LacZ plasmid was co-transfected to normalize for transfection efficiency. Results depict the mean of three experiments. Unpaired Student's t-Test assuming equal variance concluded that the increase of PCNA-luc reporter activity in RBF1, Hpo and Wts depleted cells was statistically significant from the NS control. RBF1 and Hpo depleted cells had a p-Value <0.001. Wts depleted cells had a p-Value <0.03.