PROJECT SUMMARY/ABSTRACT Inactivation of the retinoblastoma (Rb) tumor suppressor pathway is an early obligatory event in human cancer. One of the key targets of the Rb pathway is the family of E2F transcription factors, which regulate cell proliferation and apoptosis. Overlapping and redundant functions of mammalian members of the E2F and Rb tumor suppressor protein (pRB) families is a major hurdle in identification of their in vivo function. Model organisms such as Drosophila provide attractive alternatives to complement studies in mammalian systems due to high conservation of the Rb pathway and the amenability of flies to genetic analysis. Our research program is aimed at addressing two fundamental questions: what are the essential functions of the Rb pathway in development, and how do Rb pathway mutations affect different cell types in the tissue? To address these questions, we are using genetic, genomic and single-cell RNA-Seq approaches. We have identified adult skeletal muscle where E2F and Rb proteins regulate growth and myofibrillogenesis, to be important for viability. We will determine how the Rb pathway regulates gene expression and cellular metabolism during muscle development, and will investigate the systemic effects inferred by E2F deficiency in specific tissues such as adult skeletal muscle and the fat body. We have adapted Drop-Seq, a single-cell RNA-sequencing technology, to determine the effect of Rb pathway mutations in heterogeneous tissue. We have established the feasibility of this approach by building the first cell atlas of the Drosophila wild-type eye imaginal disc. Profiling of Rb mutants by Drop-Seq revealed elevated intracellular acidification in a small cell population that makes them uniquely sensitive to apoptosis. The link between apoptosis and acidification will be evaluated further. Among other questions to be addressed with Drop-seq is the investigation of why inactivation of the Hippo pathway induces dedifferentiation of Rb mutant photoreceptors. Collectively, the proposed research will enhance our knowledge of pRB and its functions during animal development.