The role of YAP/TAZ and Hippo signaling in mouse incisor stem cells

描述(由申请人提供)：啮齿动物切牙为研究干细胞提供了一个极好的模型系统，因为它可以持续生长，并从活跃的成体干细胞池中产生所有必要的细胞类型。利用小鼠遗传学，科学家已经开始了解这些细胞是如何受到不同信号通路的调控的。然而，控制它们增殖和分化的确切机制还需要进一步研究。特别是，鉴于最近的体外实验表明干细胞的行为可以被改变 在机械力的作用下，切牙干细胞也同样受到其生理环境的调节，这一点目前还知之甚少。目的/假设：在其他系统中，Hippo信号通路已被证明在整个器官水平上调节增殖和分化。重要的是，至少在细胞培养中，该途径的下游效应蛋白YAP和Tafazzin(TAZ)可以介导机械信号，以不依赖于河马的方式控制增殖和分化。因此，我们假设河马信号在切牙干细胞中以细胞自主的方式作用，决定其从增殖到分化的转变，并且YAP/TAZ还介导机械信号来调节干细胞的行为。研究设计：通过基因芯片和免疫组织化学染色，初步结果显示河马成分在切牙干细胞中表达。然而，对它们的空间表达的详细描述仍有待进行，并将在目标1中通过进行原位杂交和免疫染色以及通过构建YAP活性报告鼠来解决。在目标2中，拟议的项目将通过使用Sox2-CRE移除途径的关键组件MST1/2或SAV，来询问河马信号在切牙干细胞群体中的细胞自主功能。如果观察到一种表型，YAP将在突变背景中类似地被消融，以确定它是否作用于途径的下游。此外，这一建议的独特优势在于将小鼠遗传学与生物力学方法相结合，研究YAP/TAZ如何传递机械信号来调节干细胞行为。在目标3中，将使用原子力显微镜构建小鼠切牙的组织刚度图。这些值将用于浇注不同硬度的水凝胶，在其上培养新鲜收获的野生型或YAP/TAZ突变切牙干细胞，并研究其对机械信号的反应。与健康相关：由于不受控制的细胞生长可能导致癌症，因此在基于干细胞的治疗中是不可取的，这项多学科研究的成功完成将为开发培养策略提供遗传和生物力学目标，以获得和维持牙齿干细胞，这些干细胞可用于制造临床上安全的替换牙齿，用于治疗牙齿缺失患者。

DESCRIPTION (provided by applicant): The rodent incisor provides an excellent model system for studying stem cells because it grows continuously and generates all the necessary cell types from an active pool of adult stem cells. Using mouse genetics, scientists have begun to understand how these cells are regulated by different signaling pathways. However, the exact mechanism that controls their proliferation and differentiation requires further studies. In particular, in light of recent in vitro experiments showing that stem cell behavior can be modified by mechanical force, it is plausible that incisor stem cells are similarly regulated by their physial environment, an area that is currently poorly understood. Objective/hypothesis: The Hippo signaling pathway has been shown to regulate proliferation and differentiation at a whole organ level in other systems. Importantly, at least in cell culture, the downstream effectors of the pathway, Yes-associated Protein (YAP) and Tafazzin (TAZ), can mediate mechanical signals to control proliferation and differentiation in a Hippo-independent fashion. Therefore, we hypothesize that Hippo signaling acts cell-autonomously in the incisor stem cells to determine their transition from proliferation to differentiation and YAP/TAZ additionally mediate mechanical cues to regulate stem cell behavior. Study design: By means of microarray and immunostaining, preliminary results show that Hippo components are expressed in the incisor stem cells. However, a detailed description of their spatial expression remains to be conducted and will be addressed in Aim 1 by performing in situ hybridization and immunostaining, as well as by constructing a YAP activity reporter mouse. In Aim 2, the proposed project will interrogate the cell-autonomous function of Hippo signaling specifically in the incisor stem cell population by removing MST1/2 or SAV, key components of the pathway, using Sox2-Cre. If a phenotype is observed, YAP will be similarly ablated in the mutant background to determine if it acts downstream of the pathway. Furthermore, the unique strength of this proposal lies in combining mouse genetics with biomechanical approaches to study how YAP/TAZ may relay mechanical signals to regulate stem cell behavior. In Aim 3, a tissue stiffness map of the mouse incisor will be constructed by using atomic force microscopy. These values will be used to cast hydrogels with different stiffness, on which freshly harvested wild type or YAP/TAZ mutant incisor stem cells will be cultured and investigated for their responses to mechanical signals. Health relatedness: As uncontrolled cell growth can lead to cancer and is therefore undesirable in stem cell-based therapies, successful completion of this multidisciplinary study will provide both genetic and biomechanical targets for the development of culturing strategies to derive and maintain dental stem cells that can be used to make replacement teeth that are clinically safe for treating patients with tooth loss.