An investigation of the roles of mechanical signaling in YAP-mediated tooth renew

机械信号在 YAP 介导的牙齿更新中作用的研究

• 批准号: 9904599
• 负责人: Jimmy Kuang-Hsien Hu
• 金额: $ 24.9万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9904599
• 关键词: 3-Dimensional; Ablation; Actins; Actomyosin; Address; Adult; Advisory Committees; Affect; Area; Binding; Biology; Biomechanics; CDC42 gene; Cell Compartmentation; Cell Culture Techniques; Cell Differentiation process; Cell Proliferation; Cell Shape; Cell physiology; Cells; Cellular biology; Cervical; Chemicals; Chronic; Clinical; Collaborations; Color; Communities; Community Medicine; Culture Techniques; Data; Dental; Developmental Biology; Educational workshop; Epithelial; Epithelium; Extracellular Matrix; Extracellular Matrix Proteins; Focal Adhesion Kinase 1; Fostering; Foundations; Future; Genetic; Homeostasis; Human; Image; In Vitro; Incisor; Integrin Signaling Pathway; Integrins; Investigation; Knowledge; Laboratories; Lasers; Lead; Learning; Life Style; Light; Limb structure; Measures; Mechanics; Mediating; Mentors; Morphogenesis; Mus; Muscle Development; Mutant Strains Mice; Myosin Type II; Natural regeneration; Nonmuscle Myosin Type IIA; Nuclear; Organ; Pathway interactions; Patients; Pattern; Phase; Phosphotransferases; Play; Postdoctoral Fellow; Process; Proliferating; Proteins; Publishing; Regenerative Medicine; Regulation; Research; Research Personnel; Resources; Rodent; Role; Science; Signal Transduction; Solid; Spatial Distribution; Surveys; System; Techniques; Testing; Time; Tissues; Tooth Loss; Tooth structure; Training; Transcription Coactivator; Universities; Work; Writing; adult stem cell; base; blebbistatin; cell behavior; craniofacial; craniofacial structure; epithelial stem cell; experience; experimental study; gain of function mutation; high throughput screening; in vivo; interest; mechanotransduction; meetings; mouse genetics; non-muscle myosin; novel; progenitor; public health relevance; rho GTP-Binding Proteins; screening; self-renewal; skills; small molecule; stem cell biology; stem cell niche; stem cell proliferation; stem cells; three dimensional cell culture; two photon microscopy

 DESCRIPTION (provided by applicant): Dr. Hu is determined to become an independent researcher to investigate the mechanisms that regulate the morphogenesis and homeostasis of craniofacial structures and to apply that knowledge for translational purposes. CANDIDATE BACKGROUND: Dr. Hu did his graduate research in Dr. Clifford Tabin's laboratory at Harvard University, where he studied limb patterning and muscle development, and gained experiences in developmental biology techniques, mouse genetics, live imaging, and cell culture. Dr. Hu then joined Dr. Ophir Klein's laboratory at UCSF as a postdoctoral fellow to study the mechanisms that regulate mouse incisor stem cells, a great system for understanding stem cell-based renewal. In particular, he found YAP/TAZ as important factors for controlling stem cell proliferation and differentiation and his preliminary data suggest that mechanical signaling plays a key role in regulating YAP activity and stem cell biology. During this time, Dr. Hu gained knowledge in craniofacial and stem cell biology, and has embarked on using the incisor as a system to study mechanotransduction, while developing techniques for culturing, imaging, and measuring force. RESEARCH PLAN: Dr. Hu hypothesizes that nuclear YAP localization and YAP-mediated cellular processes are initially inhibited by actomyosin tension in the incisor stem cells and later upregulatd by integrin/FAK signaling in the transit-amplifying cells. Aim 1 (K99 portion) studies the role of cellular tension by first characterizing cellular features affected by tension, such as cell shapes, levels of phospho-Myosin II, and actin distribution in the stem cell compartment. The effects of tissue tension on cell biology and YAP localization will be examined by laser ablation, and conditional deletion of Myosin IIA/B. Aim 2 (R00 portion) focuses on integrin/FAK signaling. First, the ECM compositions in the stem cell niche will be characterized. Functional studies will then be carried out by testing the ability of ECM proteins t regulate YAP activity and cell differentiation in a 3D culture system, as well as by examining two mouse mutants, one with FAK deletion and the other with a dominant active integrin β1. Finally, a chemical screen will be performed to identify novel factors and pathways that regulate YAP and stem cell biology. Together, these Aims will address key questions in both the Hippo and stem cell field. TRAINING: Dr. Hu will learn several new techniques during the mentored phase, including laser ablation, 3D culturing, mouse knock-ins, and high throughput small molecule screening. He will also attend courses relevant to the study. In addition, Dr. Hu has assembled an advisory committee to help complete and evaluate the project. Finally, he will continue to develop his writing, presentation, managing, and mentoring skills in order to become an independent researcher. ENVIRONMENT: There are many resources and researchers in different fields at UCSF and the greater Bay area science community that are available to Dr. Hu. There are also many regular seminars, workshops, and meetings at UCSF that foster scientific interactions, sharing of ideas, collaborations, and learning opportunities. HEALTH RELATEDNESS: As adult humans don't have dental epithelial stem cells, completion of this study will provide genetic, biomechanical, and chemical targets for developing strategies to derive and maintain clinically safe dental stem cells that can be used to make replacement teeth for treating patients with tooth loss.

 描述（由申请人提供）：胡博士决心成为一名独立研究员，研究调节颅面结构的形态发生和稳态的机制，并将这些知识应用于转化目的。 Clifford Tabin 博士在哈佛大学的实验室，研究肢体模式和肌肉发育，并获得了发育生物学技术、小鼠遗传学、实时成像和细胞培养方面的经验。随后，胡博士加入加州大学旧金山分校 Ophir Klein 博士的实验室，担任博士后研究员，研究调节小鼠门牙干细胞的机制，这是一个了解干细胞更新的重要系统。特别是，他发现 Y​​AP/TAZ 是控制干细胞的重要因素。细胞增殖和分化以及他的初步数据表明机械信号在调节 YAP 活性和干细胞生物学方面发挥着关键作用。在此期间，胡博士获得了颅面和干细胞生物学方面的知识，并开始使用门牙作为研究对象。系统研究机械转导，同时开发培养、成像和测量力的技术。 研究计划：胡博士利用核 YAP 定位和 YAP 介导的细胞过程最初被切牙干细胞中的肌动球蛋白张力抑制，随后被整合素 / 上调。转运放大细胞中的 FAK 信号传导 Aim 1（K99 部分）通过首先表征受细胞张力影响的细胞特征来研究细胞张力的作用。 张力，例如细胞形状、磷酸肌球蛋白 II 的水平和干细胞区室中的肌动蛋白分布。张力组织对细胞生物学和 YAP 定位的影响将通过激光消融和肌球蛋白 IIA/B 的条件删除来检查。目标 2（R00 部分）重点关注整合素/FAK 信号传导，然后通过测试 ECM 蛋白的能力来表征干细胞生态位中的 ECM 成分。在 3D 培养系统中调节 YAP 活性和细胞分化，并通过检查两种小鼠突变体，一种具有 FAK 缺失，另一种具有显性活性整联蛋白 β1。最后，将进行化学筛选以识别新的因子和途径。这些目标将共同解决 Hippo 和干细胞领域的关键问题。 培训：胡博士将在指导阶段学习多种新技术，包括激光消融、3During cult、小鼠。他还将参加与研究敲入和高通量小分子筛选相关的课程。此外，胡博士还组建了一个顾问委员会来帮助完成和评估该项目。最后，他将继续发展他的写作和演示。环境：加州大学旧金山分校和大湾区科学界有许多不同领域的资源和研究人员可供胡博士使用，还有许多定期的研讨会、讲习班。 ，以及加州大学旧金山分校的会议，促进科学互动、分享想法、合作和学习机会：由于成年人没有牙齿上皮干细胞，这项研究的完成将为制定策略提供遗传、生物力学和化学目标，以衍生和维持临床安全。牙齿干细胞可用于制造替换牙齿，以治疗牙齿缺失的患者。