Cellular and molecular regulation of airway multiciliated cell specification and differentiation

项目摘要/摘要 肺为我们提供了将氧气从空气输送到循环系统和呼吸道的途径。 上皮细胞为肺部提供了抵御吸入病原体的第一道防线。肺上皮细胞包括 三种主要细胞类型：基础干细胞(BCS)、分泌细胞(SCs)和多纤毛细胞(MCC)。这个 这些细胞类型中每一种的比例都受到严格控制，对呼吸道功能至关重要。在疾病中，如 哮喘和慢性阻塞性肺疾病(COPD)，干细胞的一种亚型，杯状细胞，在 MCC的费用导致粘液的过度生产和无法清除病原体。虽然MCC很关键 对于呼吸道功能，从BCS产生MCC的细胞和分子步骤仍不清楚。我的 初步的单细胞RNA测序数据已经确定了BCS和MCC之间的中间细胞类型 以转录因子MyCL为标志。在这项提案中，我将回答四个问题，以解决以下主要差距 呼吸道生物学1)MyCL位于已知的早期MCC调节因子的上游还是下游？我将使用量化 细胞分辨荧光原位杂交法和免疫组化法检测血管内皮生长因子的表达 MyCL和早期的MCC监管机构。此外，我将测试MyCL+中间细胞的扩增或减少 在Notch信号的扰动下，SC和MCC的主要调节者命运。2)MyCL+中间细胞是否是 人口过境放大？我将结合原位杂交和BrdU检测来确定细胞的增殖 MyCL+中间细胞的状态。3)MyCL在MCC分化和纤毛发生中的作用是什么？我 将利用CRISPR/Cas9基因敲除技术在体外呼吸道培养系统中鉴定其功能作用 Myc1基因在MCC分化和纤毛发生中的作用。4)BCS何时承诺生产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 以及数以千计的BC的转录档案。我将鉴定BC亚型的克隆关系以确定 MCC规范的精确时序。我推测MyCL是最早的转录产物之一 MCC规范的调节者，并标志着由Notch信号调节的增殖群。 此外，我假设MyCL+中间细胞是BCS的一个亚型，注定要走向MCC 血统。这些结果将构建MCC与BCS的区别路线图，从长远来看，可能会有所帮助 确定在呼吸道疾病过程中导致不正确细胞命运决定的机制。在这项提案中，我将 接受纤毛和呼吸道生物学、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。在我的导师杰里米的专家建议和指导下 瑞特，加州大学旧金山分校内多个核心和部门的大量资源，以及与Change的合作 扎克伯格生物中心，我将做好准备，解决长期存在的呼吸道分化问题。穿过 加州大学旧金山分校提供的多个职业发展计划和杰里米·赖特的指导，我将开发这两个 成功开展独立研究项目所需的科学和实验室管理技能。

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.