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

YAP/TAZ 和 Hippo 信号在小鼠门牙干细胞中的作用

• 批准号: 8595111
• 负责人: Jimmy Kuang-Hsien Hu
• 金额: $ 5万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8595111
• 关键词: Address; Adult; Affect; Area; Atomic Force Microscopy; Biological Models; Biology; Biomechanics; Cell Culture System; Cell Culture Techniques; Cell Differentiation process; Cell Proliferation; Cell Therapy; Cells; Cellular biology; Cervical; Chronic; Communities; Cues; Data; Dental; Dental caries; Development; Disinhibition; Environment; Epithelial; Excision; Generations; Genes; Genetic; Harvest; Health; Human; Hydrogels; In Situ Hybridization; In Vitro; Incisor; Laboratory Finding; Lead; Life Style; Light; Malignant Neoplasms; Maps; Measures; Mechanics; Mediating; Methods; Mus; Natural regeneration; Neighborhoods; Organ; Pathway interactions; Patients; Pattern; Phenotype; Physical environment; Property; Proteins; Reading; Regenerative Medicine; Regulation; Reporter; Research Design; Rodent; Role; Route; Sampling; Scientist; Signal Pathway; Signal Transduction; Stem cells; System; Techniques; Testing; Tissues; Tooth Loss; Tooth structure; Translating; Travel; adult stem cell; cell behavior; cell type; clinical application; craniofacial; interest; multidisciplinary; mutant; public health relevance; research study; response; stem; stem cell niche; stem cell population; tool; two-dimensional; uncontrolled cell growth

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.

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，因为它连续生长，并从活跃的成体干细胞库中产生所有必需的细胞类型。利用小鼠遗传学，科学家们已经开始了解这些细胞如何受到不同信号通路的调节。然而，控制其增殖和分化的确切机制需要进一步研究。特别是，鉴于最近的体外实验表明，干细胞的行为可以改变， 通过机械力，门齿干细胞类似地受到其生理环境的调节是合理的，这是目前知之甚少的领域。目的/假设：Hippo信号通路已被证明在其他系统中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，该途径的下游效应物，即Yes相关蛋白（雅普）和Tafazzin（TAZ），可以介导机械信号以Hippo非依赖性方式控制增殖和分化。因此，我们假设Hippo信号传导在切牙干细胞中自主地起作用以确定它们从增殖到分化的转变，并且雅普/TAZ另外介导机械信号以调节干细胞行为。研究设计：通过微阵列和免疫染色的方法，初步结果显示Hippo成分在切牙干细胞中表达。然而，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雅普活性报告小鼠来解决。在目标2中，拟议的项目将通过使用Sox2-Cre去除MST 1/2或SAV（途径的关键组分）来询问Hippo信号传导的细胞自主功能，特别是在切牙干细胞群中。如果观察到表型，则将在突变体背景中类似地消融雅普，以确定其是否在途径下游起作用。此外，该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，以研究雅普/TAZ如何传递机械信号来调节干细胞行为。在目标3中，将通过使用原子力显微镜构建小鼠切牙的组织刚度图。这些值将用于浇铸具有不同硬度的水凝胶，在其上培养新鲜收获的野生型或雅普/TAZ突变体切牙干细胞并研究它们对机械信号的响应。健康相关性：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培养策略的发展提供遗传和生物力学目标，以获得和维持可用于制造临床安全治疗牙齿缺失患者的替代牙齿的牙科干细胞。