Elucidating cellular competition in mammalian epithelium and the role of Myh9.

我计划建立一个研究项目，该项目将在其天然生理环境中对上皮生物学的功能研究采取全面和定量的方法。我的目标是确定在成人稳态、癌症和慢性以及癌症相关炎症中调节上皮组织生长的分子和细胞机制。对于NSERC发现资助项目，我建议阐明组织驻留干细胞的细胞竞争，细胞骨架成分如肌球蛋白IIa和DNA损伤修复之间的密切关系。 此外，我们将建立和完善我们的体内CRISPR基因组编辑能力，以在发育和组织稳态过程中对完整的上皮进行遗传操作。该项目围绕生物学中的一个基本问题，即干细胞如何平衡自我更新，分化和组织稳态。由于成体干细胞是各种谱系特异性细胞的终身储存库，因此它们对自发性DNA损伤特别敏感。影响其自我更新或分化能力的突变可能会导致组织萎缩（器官退化）并因干细胞耗竭而加速衰老，而导致增殖优势的突变可能导致克隆扩增甚至细胞竞争。DNA损伤反应的中心是p53通路，它作为中继来感知损伤，并触发细胞周期停滞以进行DNA修复或触发细胞死亡。有趣的是，我们最近发现肌球蛋白IIa，肌动蛋白细胞骨架的一个组成部分，是绝对需要的DDR诱导的p53激活-然而，确切的分子机制仍然难以捉摸。因此，我们计划进行一组生化和蛋白质组学实验，以阐明确切的分子机制参与肌球蛋白IIa-p53的相互作用。此外，我们将建立我们独特的体内RNAi注射方法，并建立新的直接体内CRISPR基因组编辑能力，以研究肌球蛋白IIa在发育和成人稳态过程中的干细胞行为和细胞竞争，使用小鼠皮肤表皮作为我们的模型系统。利用这项技术，我们将开发一种系统生物学筛选方法，这将使我们能够在哺乳动物器官系统中首次筛选和表征控制细胞竞争的基本因素。重要的是，我们的体内方法规避了常规细胞培养筛选的所有限制，并评估了嵌入免疫活性小鼠生理组织环境中的细胞中的基因功能，这对于严重依赖于基质线索和生态位信号的干细胞和祖细胞尤其重要。从这些研究中获得的技术专长肯定也将在我们的其他项目中得到利用，并将成为一个丰富的合作平台的基础。**

I plan to establish a research program that will stand out for taking a comprehensive and quantitative approach to functional studies in epithelial biology in its native, physiological environment. My goal is to identify molecular and cellular mechanisms that regulate epithelial tissue growth in adult homeostasis, cancer and chronic as well as cancer-associated inflammation. For the NSERC Discovery Grant project, I am proposing to elucidate the intimate relationship between cellular competition of tissue resident stem cells, cytoskeletal components such as myosin IIa and DNA damage repair. In addition, we will establish and refine our in vivo CRISPR genome-editing capabilities to genetically manipulate an intact epithelium during development and tissue homeostasis.***This project centers around one of the fundamental questions in biology, namely how stem cells balance self-renewal, differentiation and tissue homeostasis. As adult stem cells are a life-time reservoir for various lineage-specific cells, they are especially sensitive to spontaneous DNA damage. Mutations that impinge on their self-renewal or differentiation capacity may lead to tissue atrophy (organ degeneration) and accelerate aging due to stem cell exhaustion, while mutations resulting in a proliferative advantage can result clonal expansion and even cell competition. Central to the DNA damage response is the p53 pathway, which acts as relay to sense the damage and either triggers cell cycle arrest for DNA repair to happen or triggers cell death. Interestingly, we recently identified myosin IIa, an integral part of the actin-cytoskeleton, to be absolutely required for DDR-induced p53 activation – however; the exact molecular mechanism remains elusive. We therefore plan to perform a panel of biochemical and proteomics experiments to elucidate the exact molecular machinery involved in myosinIIa-p53 interaction. Additionally, we will build upon our unique in vivo RNAi injection methodology and establish novel direct in vivo CRISPR genome-editing capabilities to study myosin IIa in stem cell behavior and cellular competition during development and adult homeostasis using mouse skin epidermis as our model system. Using this technology, we will then develop a Systems Biology screening approach, which will allow us to screen for and characterize essential factors governing cell competition for the first time in a mammalian organ system.***Importantly, our in vivo approach circumvents all limitations of conventional cell culture screens and assesses gene function in cells embedded within physiological tissue environment of immune-competent mice, which is paramount esp. for stem and progenitor cells, which heavily depend on stromal cues and niche signals. The technical expertise gained from those studies will certainly also be leveraged in our other projects and will be the basis for a rich collaborative platform. **