Abstract Our prior studies have suggested that genes mapping around the tumor suppressor RB1 termed forerunner (FR) genes were silenced by methylation or mutations in in situ phases of urothelial carcinogenesis and were potentially driving the development of clonal mucosal field effects. In order to verify these correlative observations, we have developed a mouse ablation model of a candidate FR gene, P2RY5 mapping within intron 17 of RB1 and encoding G protein receptor. In order to facilitate a germline ablation of P2RY5, a knock-out construct was generated to delete the P2RY5 reading frame. The construct was inserted into mouse embryonic stem cells and the gene was ablated by mating the animals with CMV-cre mice. The effect of P2RY5 deletion on cell proliferation was assessed on cultured fibroblasts derived from embryos with P2RY5 ablation (P2RY5-/-) and from control embryos (P2RY5 +/+). The effect of P2RY5 ablation in the urothelium was assessed on bladders of P2RY5-/- and P2RY5 +/+ mice. The bladders were fixed in formalin and the proliferation status in the urothelium was assessed by immunohistochemical and immunoflourescent staining for Ki67and PCNA. The status of the urothelium was assessed by immunohistochemical and immunoflourescent staining for cytokeratins 5, 14, and 18. In addition, the effect of silencing P2RY5 on global gene expression patterns was evaluated on mouse embryonic fibroblasts using Illumina mouse cDNA expression microarrays. P2RY5-/- embryonic fibroblasts showed dramatically increased proliferation and colony formation rates as compared to P2RY5 +/+ cells. cDNA micro analysis shows that silencing P2RY5 activated several important oncogenic pathways including NFκB and STAT3. Similarly, P2RY5-/- urothelium contained nearly three times more proliferating cells as compared to P2RY5 +/+ urothelium. The increased proliferation rate was associated with the striking expansion of the peribasal layer as documented by staining for cytokeratin 5 and 14. In the P2RY5 +/+ urothelium the cytokeratin 5 and 14 positive cells formed a sparse single basal layer. In contrast, in the P2RY5-/- urothelium, these cells formed irregular mounds overcrowded with peribasal cells. Taken together, our observations show that the silencing of P2RY5 increases the growth rate of mouse embryonic fibroblasts via the activation of several oncogenic pathways and causes an increase in the proliferation rate in the urothelium resulting in urothelial hyperplasia recapitulating incipient events of urothelial carcinogenisis. Citation Format: Bogdan A. Czerniak, Sangkyou Lee, Ying Wang, Jolanta Bondaruk, Taddeusz Majewski, Woongyoung Choi, David McConkey, Richard R. Behringer. Germline ablation of candidate forerunner gene, P2RY5, in a mouse model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 307. doi:10.1158/1538-7445.AM2013-307