To define the role of SOX9 and Sox9+ cells in alveolar homeostasis and regeneration

 描述（由申请人提供）：肺上皮易于因吸入病原体和污染物而频繁和复发性损伤。因此，了解肺泡干细胞和祖细胞的再生能力和作用具有相当大的实际意义和治疗意义。受损肺泡的修复对于恢复正常肺功能至关重要，但调节修复的细胞和分子机制仍不清楚。我们已经确定了一种新的肺干/祖细胞群体在远端肺泡标记的Sox 9，这些细胞是解剖学和分子不同于已知的肺泡祖细胞。我们还发现，表达Sox 9的细胞有助于流感损伤后的远端肺泡，并且损伤后Sox 9蛋白的表达增加。基于这些初步数据，我们假设这些新的Sox 9+细胞是肺泡的多能可塑性祖细胞，对于气道和肺泡损伤修复至关重要。我们还假设转录因子SOX 9通过与关键的肺特异性转录因子合作来调节成人肺泡内稳态和再生，从而调节损伤修复。为了验证这些假设，我们提出了以下具体目标。在K99阶段，我们将1）确定一种新的Sox 9表达细胞群在肺泡稳态和肺泡损伤修复中的作用; 2）确定SOX 9功能的获得和丧失对成人肺泡稳态和再生的影响。在本申请的ROO阶段，我们将定义SOX 9调节成人肺泡维持和再生的分子机制。我们将同时使用谱系追踪和细胞消融结合流感损伤的小鼠模型来研究Sox 9+细胞对再生肺的贡献。我们还将分别使用条件性异位表达和功能丧失小鼠研究Sox 9获得和功能丧失的影响。最后，我们将确定Sox 9相互作用的蛋白质和Sox 9结合的基因组位点，分别使用免疫共沉淀和染色质IP实验，然后对靶蛋白和基因进行功能分析。我们还将讨论这些新细胞和分子机制的激活是否加速肺泡修复，使这些细胞具有治疗意义。我是马萨诸塞州总医院再生医学中心的博士后研究员。从我的硕士，博士。通过博士后培训，我在干细胞和分子生物学方面获得了丰富的研究经验。我在开发新的小鼠损伤模型，鉴定新基因和新的遗传和表观遗传转录调控元件方面获得了大量的专业知识。近年来，我已经开发了新的工具，用于调节肺组织特异性基因表达，并确定了一个显着的细胞可塑性现象，在再生气道，其中分化的细胞可以成为干细胞损伤后。在目前的建议中，我寻求支持，研究一种新的假定的干细胞群和相关的转录和信号转导通路在肺泡。我打算从我的导师Jayaraj Rajagopal博士和共同导师大卫斯卡登博士那里获得科学和职业发展技能。Rajagopal博士拥有所有必要的干细胞和小鼠遗传学专业知识，以及Sox蛋白和Wnt信号的经验，以指导我的早期研究计划，我已经开发了一些专业知识来处理本赠款R 00阶段提出的生化研究。我选择Rajagopal博士的实验室，因为他被认为是肺干细胞生物学的新星之一。但为了有更广泛的指导，我将利用大卫斯卡登博士的专业知识，他是著名的干细胞生物学家，也是哈佛干细胞研究所的主任，他自己培养了许多独立的PI的职业生涯，康拉德霍切德林格博士，转录调控和Sox蛋白的专家，惠灵顿卡多佐博士，气道和肺特异性信号通路的专家，以及肺泡生物学专家达雷尔·科顿博士马萨诸塞州总医院、哈佛干细胞研究所、哈佛大学和哈佛医学院丰富的研究环境和设施，以及我的导师和顾问成员的专家指导，将帮助我发展新的技能，成为一名独立的研究者。我将把我的100%的时间专注于研究计划和培训活动，包括出席和演示在科学和职业发展活动。

 DESCRIPTION (provided by applicant): Lung epithelium is prone to frequent and recurrent injuries from inhaled pathogens and pollutants. Understanding the regenerative capacity and the role of resident alveolar stem and progenitor cells is therefore of considerable practical and therapeutic interest. Repair of damaged alveoli is crucial for restoration of normal lung function but the cellular and molecular mechanisms regulating repair are still not well understood. We have identified a novel lung stem/progenitor population in the distal alveoli that is marked by Sox9 and that these cells are anatomically and molecularly distinct from the known alveolar progenitor cells. We also find that Sox9 expressing cells contribute to the distal alveoli followin influenza injury and that the expression of SOX9 protein us increased following injury. Based on this preliminary data, we hypothesize that these novel Sox9+ cells are multipotent plastic progenitor cells of the alveoli that are essential for airway and alveolar injury repair. We also hypothesize that the transcription factor SOX9 is required to regulate injury repair by co-operating with key lung-specific transcription factor to regulate adult alveolar homeostasis and regeneration. To test these hypotheses, we propose the following specific aims. In the K99 phase of the proposal, we will 1) determine the role of a novel population of Sox9-expressing cells in alveolar homeostasis and in alveolar injury repair; 2) determine the effect of gain and loss of function of SOX9 on adult alveolar homeostasis and regeneration. In the ROO phase of this application, we will define the molecular mechanisms through which SOX9 regulates adult alveolar maintenance and regeneration. We will simultaneously use lineage tracing and cell ablation murine models in combination with influenza injury to study the contribution of Sox9+ cells to regenerated lung. We will also study the effects of Sox9 gain and loss of function using conditional ectopic expression and loss of function mice, respectively. Finally, we will identify Sox9 interacting proteins and Sox9 bound genomic loci using co-immunoprecipitation and Chromatin IP experiments, respectively, followed by functional analysis of target proteins and genes. We will also address the question whether activation of these novel cells and molecular mechanisms accelerates alveolar repair, making these cells therapeutically relevant. I am a postdoctoral fellow at the Center for Regenerative Medicine, Massachusetts General Hospital. From my masters, Ph.D. and current postdoctoral training I have gained substantial research experience in stem cell and molecular biology. I have gained substantial expertise in developing new mouse models of injury, identifying novel genes and novel genetic and epigenetic transcription regulatory elements. In recent years, I have developed novel tools for modulating gene expression specific to lung tissues and identified a remarkable cellular plasticity phenomenon in regenerating airway in which differentiated cells can become stem cells after injury. In the current proposal I seek support to study a novel putative stem cell population and the associated transcriptional and signaling pathways in the alveoli. I intend to gain both scientific and career development skills from my mentor Dr. Jayaraj Rajagopal and co-mentor Dr. David Scadden. Dr. Rajagopal has all the necessary stem cell and mouse genetics expertise as well as the experience with Sox proteins and Wnt signaling to guide my early research plan and I have already developed some of the expertise to handle the biochemical studies proposed in the R00 phase of this grant. I chose Dr. Rajagopal's lab since he is considered one of the rising stars in lung stem cell biology. But in the interests of having a breadth of mentorship, I wll take advantage of the expertise from of Dr. David Scadden, a renowned stem cell biologist and director of the Harvard Stem Cell Institute that himself has fostered the career of many independent PIs, Dr. Konrad Hochedlinger, an expert in transcriptional regulation and Sox-proteins, Dr. Wellington Cardoso, an expert in airway and lung specific signaling pathways, and Dr. Darrel Kotton, an expert in alveolar biology. The rich research environment and facilities at the Massachusetts General Hospital, Harvard Stem Cell Institute, Harvard University, and Harvard Medical School and the expert guidance from my mentors and advisory members will help me to develop new skills to establish as an independent investigator. I will devote my 100% of my time to focused research program and to training activities including attendance and presentation at both scientific and career developmental activities.