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

 描述（由申请人提供）：胡博士决心成为一名独立的研究人员，研究调节颅面结构形态发生和稳态的机制，并将这些知识用于翻译目的。候选人背景：胡博士在哈佛大学Clifford Tabin博士的实验室进行了研究生研究，在那里他研究了肢体模式和肌肉发育，并在发育生物学技术、小鼠遗传学、活体成像和细胞培养方面积累了经验。然后，胡博士加入了UCSF的Ophir Klein博士的实验室，担任博士后研究员，研究调节小鼠切牙干细胞的机制，这是一个了解基于干细胞的更新的伟大系统。特别是，他发现雅普/TAZ是控制干细胞增殖和分化的重要因素，他的初步数据表明，机械信号在调节雅普活性和干细胞生物学中起着关键作用。在此期间，胡博士获得了颅面和干细胞生物学方面的知识，并开始使用切牙作为一个系统来研究机械转导，同时开发培养，成像和测量力的技术。研究报告：Hu博士推测，细胞核雅普定位和YAP介导的细胞过程最初受到切牙干细胞中肌动球蛋白张力的抑制，随后受到传递放大细胞中整合素/FAK信号的上调。目的1（K99部分）通过首先表征受以下因素影响的细胞特征来研究细胞张力的作用： 张力，如细胞形状，磷酸肌球蛋白II的水平，和肌动蛋白在干细胞室中的分布。将通过激光消融和肌球蛋白IIA/B的条件性缺失检查组织张力对细胞生物学和雅普定位的影响。目标2（R 00部分）集中于整合素/FAK信号传导。首先，将表征干细胞龛中的ECM组合物。然后通过测试ECM蛋白在3D培养系统中调节雅普活性和细胞分化的能力，以及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合素β1）来进行功能研究。最后，将进行化学筛选以鉴定调节雅普和干细胞生物学的新因子和途径。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几项新技术，包括激光消融、3D培养、小鼠基因敲入和高通量小分子筛选。他还将参加与研究有关的课程。此外，胡博士还组建了一个咨询委员会，以帮助完成和评估该项目。最后，他将继续发展他的写作，演讲，管理和指导技能，以成为一名独立的研究人员。环境：有许多资源和研究人员在不同领域在加州大学旧金山分校和大湾区科学界，可供胡博士。也有许多定期研讨会，讲习班，并在UCSF促进科学互动，思想，合作和学习机会的分享会议。健康保障：由于成年人没有牙齿上皮干细胞，这项研究的完成将为开发策略提供遗传，生物力学和化学目标，以获得和维持临床安全的牙齿干细胞，这些干细胞可用于制造替代牙齿，以治疗牙齿缺失患者。

 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.