Cellular and molecular regulation of airway multiciliated cell specification and differentiation

气道多纤毛细胞规范和分化的细胞和分子调节

• 批准号: 10409531
• 负责人: Lauren Byrnes
• 金额: $ 6.64万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10409531
• 关键词: Address; Air; Airway Disease; Asthma; Bar Codes; Basal Cell; Biological Assay; Biology; Bromodeoxyuridine; CRISPR/Cas technology; Cardiovascular system; Cell Differentiation process; Cell Lineage; Cells; Chronic Obstructive Airway Disease; Collaborations; Computer Analysis; Data; Data Set; Decision Making; Development; Disease; Epithelial; Epithelial Cells; Failure; Fluorescent in Situ Hybridization; Gene Expression; Gene Expression Profile; Genes; Genetic Transcription; Goblet Cells; Health; Hyperplasia; In Situ Hybridization; In Vitro; Inhalation; Injury; Knock-out; Knowledge; Laboratories; Lung; Lung diseases; Maps; Measures; Mediating; Mentors; Mentorship; Methodology; Molecular; Molecular Profiling; Mucous body substance; Mutagenesis; Oxygen; Pathology; Patients; Pattern; Play; Population; Production; Program Development; RNA; Regulation; Research; Resolution; Resources; Role; Running; Secretory Cell; Signal Transduction; System; Technology; Testing; Training; Work; airway epithelium; career development; cell fate specification; cell type; cilium biogenesis; experimental study; in vivo; innovation; notch protein; novel; pathogen; precursor cell; progenitor; programs; pulmonary function; single-cell RNA sequencing; skills; stem cell differentiation; stem cell fate; stem cells; transcription factor

Project Summary/Abstract Lungs provide the means by which we transfer oxygen from air to the circulatory system, and the airway epithelium provides the lungs’ first line of defense against inhaled pathogens. The lung epithelium is comprised of three major cell types: basal stem cells (BCs), secretory cells (SCs) and multiciliated cells (MCCs). The proportion of each of these cell types is tightly controlled and critical for airway function. In diseases such as asthma and chronic obstructive pulmonary disease (COPD), hyperplasia of a subtype of SCs, goblet cells, at the expense of MCCs results in an overproduction of mucus and a failure to clear pathogens. While MCCs are critical for airway function, the cellular and molecular steps that generate MCCs from BCs are still not understood. My preliminary single-cell RNA-sequencing data has identified an intermediate cell type between BCs and MCCs marked by the transcription factor, Mycl. In this proposal, I will answer four questions to address major gaps in airway biology 1) Is Mycl upstream or downstream of known early MCC regulators? I will utilize quantitative cellular resolution fluorescent in situ hybridization and immunostaining to determine the expression patterns of Mycl and early MCC regulators. Additionally, I will test the expansion or reduction of Mycl+ intermediate cells upon perturbation of Notch signaling, a major regulator of SC and MCC fates. 2) Are Mycl+ intermediate cells a transit-amplifying population? I will combine in situ hybridization with BrdU assays to determine the proliferation status of Mycl+ intermediate cells. 3) What is the function of Mycl during MCC differentiation and ciliogenesis? I will utilize CRISPR/Cas9 knockout technology in an in vitro airway culture system to identify the functional role of the gene Mycl in MCC differentiation and ciliogenesis. 4) When do BCs commit to producing MCCs or SCs? I will combine cellular barcoding and single-cell RNA-sequencing to simultaneously measure the clonal lineages and transcriptional profiles of thousands of BCs. I will identify the clonal relationships of BC subtypes to determine the precise timing of MCC specification. I hypothesize that Mycl is one of the earliest transcriptional regulators of MCC specification, and marks a proliferative population regulated by Notch signaling. Furthermore, I hypothesize that Mycl+ intermediate cells are a subtype of BCs, fated towards the MCC lineage. These results will build a roadmap of MCC differentiation from BCs, and in the longer term, may help identify the mechanisms leading to improper cell fate decisions during airway disease. During this proposal, I will receive training in ciliary and airway biology, single-cell RNA-sequencing methodology, and computational analysis of single-cell RNA-sequencing datasets. With expert advice and guidance from my mentor, Jeremy Reiter, the vast resources of multiple cores and departments within UCSF, and a collaboration with the Chan Zuckerberg Biohub, I will be well-equipped to tackle longstanding questions of airway differentiation. Through multiple career development programs offered by UCSF and Jeremy Reiter’s mentorship, I will develop both the scientific and laboratory management skills required to run a successful independent research program.

项目概要/摘要 肺提供了我们将空气中的氧气转移到循环系统的途径，而气道 上皮细胞是肺部抵御吸入病原体的第一道防线。肺上皮包括 三种主要细胞类型：基底干细胞（BC）、分泌细胞（SC）和多纤毛细胞（MCC）。这 这些细胞类型中每种细胞的比例都受到严格控制，并且对气道功能至关重要。在疾病中，例如 哮喘和慢性阻塞性肺病 (COPD)、SC 亚型、杯状细胞的增生 MCC 的消耗会导致粘液产生过多并且无法清除病原体。虽然 MCC 至关重要 对于气道功能，从 BC 产生 MCC 的细胞和分子步骤仍不清楚。我的 初步的单细胞 RNA 测序数据已确定了 BC 和 MCC 之间的中间细胞类型 由转录因子 Mycl 标记。在本提案中，我将回答四个问题，以解决以下方面的主要差距： 气道生物学 1) Mycl 是已知早期 MCC 调节因子的上游还是下游？我将利用定量 细胞分辨率荧光原位杂交和免疫染色以确定表达模式 Mycl 和早期 MCC 监管机构。另外，我将测试Mycl+中间细胞的扩增或缩小 Notch 信号传导受到扰动，Notch 信号是 SC 和 MCC 命运的主要调节因子。 2) Mycl+中间细胞是 交通放大人口？我将结合原位杂交和 BrdU 检测来确定增殖情况 Mycl+中间细胞的状态。 3）Mycl在MCC分化和纤毛发生过程中的功能是什么？我 将在体外气道培养系统中利用 CRISPR/Cas9 敲除技术来确定其功能作用 Mycl 基因在 MCC 分化和纤毛发生中的作用。 4) BC什么时候承诺生产MCC或SC？ 我将结合细胞条形码和单细胞 RNA 测序来同时测量克隆谱系 和数千个BC的转录谱。我将鉴定 BC 亚型的克隆关系以确定 MCC规范的精确计时。我假设 Mycl 是最早的转录因子之一 MCC 规范的调节因子，并标志着受 Notch 信号调节的增殖群体。 此外，我假设 Mycl+ 中间细胞是 BC 的一种亚型，注定走向 MCC 血统。这些结果将构建 MCC 与 BC 的区别路线图，从长远来看，可能会有所帮助 确定导致气道疾病期间细胞命运决定不当的机制。在这个提议期间，我将 接受纤毛和气道生物学、单细胞 RNA 测序方法和计算方面的培训 单细胞 RNA 测序数据集的分析。在我的导师 Jeremy 的专家建议和指导下 Reiter、UCSF 内多个核心和部门的丰富资源以及与 Chan 的合作 扎克伯格生物中心，我将有能力解决长期存在的气道分化问题。通过 在 UCSF 提供的多个职业发展计划和 Jeremy Reiter 的指导下，我将同时发展 成功开展独立研究项目所需的科学和实验室管理技能。