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

 描述(申请人提供)：肺上皮容易因吸入病原体和污染物而频繁和反复损伤。因此，了解常驻肺泡干细胞和祖细胞的再生能力和作用具有相当大的实用和治疗意义。受损肺泡的修复是恢复正常肺功能的关键，但调节修复的细胞和分子机制仍不清楚。我们在远端肺泡中发现了一个新的肺干/祖细胞群体，以SOX9为标志，这些细胞在解剖学和分子上与已知的肺泡祖细胞不同。我们还发现，SOX9表达的细胞参与了流感损伤后的远端肺泡，损伤后SOX9蛋白的表达增加。基于这些初步数据，我们推测这些新的Sox9+细胞是肺泡的多潜能可塑性前体细胞，对于呼吸道和肺泡损伤的修复是必不可少的。我们还假设，转录因子SOX9需要与关键的肺特异性转录因子协同作用来调节损伤修复，以调节成人肺泡的动态平衡和再生。为了检验这些假设，我们提出了以下具体目标。在提案的K99阶段，我们将1)确定一组新的表达Sox9的细胞在肺泡动态平衡和肺泡损伤修复中的作用；2)确定SOX9功能的获得和丧失对成人肺泡动态平衡和再生的影响。在这一应用的Roo阶段，我们将确定SOX9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小鼠模型，结合流感损伤来研究Sox9+细胞对再生肺的贡献。我们还将利用条件性异位表达和功能丧失小鼠分别研究Sox9功能获得和功能丧失的影响。最后，我们将分别通过免疫共沉淀和染色质IP实验鉴定Sox9相互作用蛋白和Sox9结合基因组位点，然后进行目标蛋白和基因的功能分析。我们还将解决这些新细胞和分子机制的激活是否加速肺泡修复的问题，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。在我的硕士、博士和目前的博士后培训中，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，识别新的基因和新的遗传和表观遗传转录调控元件方面获得了丰富的专业知识。近年来，我开发了新的工具来调节肺组织特有的基因表达，并在再生气道中发现了一种显著的细胞可塑性现象，在这种现象中，分化的细胞可以在损伤后成为干细胞。在目前的提案中，我寻求支持来研究一个新的假定的干细胞群体以及肺泡中相关的转录和信号通路。我打算从我的导师Jayaraj Rajagopal博士和共同导师David Scadden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蛋白质和Wnt信号方面的经验来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这笔赠款的R00阶段中提出的生化研究。我选择了拉贾戈帕尔博士的实验室，因为他被认为是肺干细胞生物学领域的后起之秀之一。但为了获得广泛的指导，我将利用著名干细胞生物学家、哈佛干细胞研究所所长大卫·斯卡登博士、转录调控和Sox蛋白专家康拉德·霍切德林格博士、呼吸道和肺特定信号通路专家威灵顿·卡多佐博士以及肺泡生物学专家达雷尔·科顿博士的专业知识。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，将帮助我培养成为一名独立研究员的新技能。我将把100%的时间投入到有重点的研究项目和培训活动中，包括参加科学和职业发展活动并发表演讲。

 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.