1076 VOLUME 41 | NUMBER 10 | OCTOBER 2009 NATURE GENETICS ARTICLES Coordination between cellular proliferation and differentiation controls and maintains homeostasis in multicellular organisms. Appropriate numbers of undifferentiated cells must be generated at specific developmental stages and these cells must exit the cell cycle in a tightly regulated manner to ensure proper cell fate specification and pattern formation. PcG genes encode epigenetic factors that repress developmental target genes. PcG proteins are involved in many biological processes, including repression of homeotic gene transcription, regulation of stem cell identity, genomic imprinting and X-chromosome inactiva- tion 1 . Mutation or misexpression of PcG genes has been associated with several types of human cancer. However, tumors accumulate multiple genetic and epigenetic modifications, and it has not been shown that PcG factors can cause neoplastic transformation 2 . Here, we have addressed this issue in vivo using the developing Drosophila eye imaginal disc as a model system. The larval imagi- nal discs, which share several properties with mammalian epithelial tissues, are composed of epithelial cells that must maintain proportional growth, differentiation and renewal to generate adult tissues and body structures correctly. Imaginal disc cells proliferate and differentiate in response to molecular pathways that are highly conserved across species and that are perturbed in oncogenesis. PcG proteins regulate cell cycle genes such as Cdkn2a (also known as Ink4a and Arf) in vertebrates 3,4 . Moreover, many genes that become silenced in cancer cells by DNA hypermethylation have been identified as PcG target genes in stem cells 5–7 , and the oncogene Bmi1 can control tumor development even in Cdkn2a-deficient mice 8 . This indicates that Polycomb-dependent control of cell proliferation might involve multiple mechanisms that remain to be elucidated. Likewise, PcG proteins control cell cycle genes in Drosophila, but their regulatory function is not under- stood 9–11 . Therefore, we analyzed the role of PcG genes in the regulation of cell proliferation in the Drosophila eye. RESULTS Mutation of the PcG locus ph leads to tissue overgrowth All PcG genes have an essential function in Drosophila. Therefore, we generated clones of homozygous null mutant cells in otherwise heterozygous animals by mitotic recombination using the mosaic analysis with a repressible cell marker (MARCM) system 12,13 . We used MARCM to knock out both genes (ph-p and ph-d) in the ph locus (see Online Methods) in order to eliminate the PH protein, an essen- tial stoichiometric component of the multimeric Polycomb complex PRC1 (ref. 14). To trigger mitotic recombination, we expressed FLP recombinase under the control of the early-expressed ey promoter (eyFLP). MARCM clones of ph null tissue, labeled by green fluores- cent protein (GFP; Supplementary Fig. 1) and examined at the late third-instar larval stage, showed a marked overgrowth (Fig. 1). Larvae bearing ph null clones are bigger than larvae bearing equivalent neu- tral clones owing to an extension in the duration of the larval stages (Fig. 1a), a typical phenotype of tumor suppressor gene mutations 15,16 . Moreover, larvae with ph null clones show a stronger GFP signal in the anterior body than control larvae, indicating that there might be an increase in the mutant cell mass. Eye imaginal discs containing ph null clones were up to three times larger than control eye discs Polyhomeotic has a tumor suppressor activity mediated by repression of Notch signaling Anne-Marie Martinez 1,2 , Bernd Schuettengruber 1 , Samy Sakr 1 , Ana Janic 3 , Cayetano Gonzalez 3 & Giacomo Cavalli 1 Polycomb Group (PcG) proteins silence critical developmental genes and modulate cell proliferation. Using the Drosophila melanogaster eye as a model system, we show that cells with mutations in the gene locus (ph) that encodes the PcG protein Polyhomeotic (PH) overproliferate and lose both the ability to differentiate and their normal polarity. They invade the neighboring tissues and, when combined with an activated form of the Ras proto-oncogene, they trigger the formation of metastases. PcG proteins bind to multiple genes in the Notch pathway and control their transcription as well as Notch signaling. The massive cell-autonomous overproliferation of ph mutant cell clones can be rescued by ectopic expression of a dominant negative form of Notch or by RNA interference (RNAi)-mediated repression of Notch. Conversely, overexpression of ph induces a small-eye phenotype that is rescued by activation of Notch signaling. These data show that ph is a tumor suppressor locus that controls cellular proliferation by silencing multiple Notch signaling components. 1 Institut de Génétique Humaine, Centre Nationale de la Recherche Scientifique, Montpellier, France. 2 Faculté des Sciences, Université de Montpellier 2, Montpellier, France. 3 Cell Division Group, Institute for Research in Biomedicine and Institució Catalana de Recerca i Estudis Avançats, Parc Científic Barcelona, Barcelona, Spain. Correspondence should be addressed to G.C. (Giacomo.Cavalli@igh.cnrs.fr) or A.-M.M. (Anne-Marie.Martinez@igh.cnrs.fr). Received 2 April; accepted 5 June; published online 13 September 2009; doi:10.1038/ng.414 © 2009 Nature America, Inc. All rights reserved.