Control of cell proliferation and differentiation by the Retinoblastoma tumor suppressor pathway

项目摘要/摘要 视网膜母细胞瘤(RB)肿瘤抑制通路失活是人类早期的必经事件 癌症。Rb途径的关键靶点之一是E2F转录因子家族，它调节 细胞增殖和凋亡。E2F中哺乳动物成员的重叠和冗余功能 而Rb肿瘤抑制蛋白(PRB)家族是鉴定其体内功能的主要障碍。 果蝇等模式生物为哺乳动物的补充研究提供了有吸引力的替代方案 由于RB途径的高度保守性和苍蝇对遗传分析的适应性，该系统具有很高的安全性。我们的 研究计划旨在解决两个基本问题：什么是基本功能 发育中的Rb通路，以及Rb通路突变如何影响不同类型的细胞 纸巾？为了解决这些问题，我们正在使用遗传、基因组和单细胞RNA-SEQ方法。 我们已经鉴定出成人骨骼肌中E2F和Rb蛋白调节生长和 肌原纤维生成，对生存能力很重要。我们将确定Rb通路如何调节基因 在肌肉发育过程中的表达和细胞代谢，并将调查全身的影响 由特定组织，如成人骨骼肌和脂肪体中的E2F缺乏所推断。我们有 采用Drop-Seq单细胞RNA测序技术确定Rb通路的作用 异质组织中的突变。我们已经通过构建 果蝇野生型眼表象盘的首个细胞图谱。利用Drop-Seq技术分析Rb突变体 小细胞群体中细胞内酸化水平升高，使其对 细胞凋亡。将进一步评估细胞凋亡和酸化之间的联系。在其他问题中 要用Drop-Seq解决的是为什么河马途径失活会导致 Rb突变光感受器的去分化。总的来说，拟议的研究将加强我们的 了解PRB及其在动物发育过程中的作用。

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.