Cellular and molecular regulation of airway multiciliated cell specification and differentiation

项目总结/摘要 肺提供了我们将氧气从空气中转移到循环系统的手段， 上皮细胞是肺部抵御吸入病原体的第一道防线。肺上皮由 三种主要的细胞类型：基底干细胞（BC），分泌细胞（SC）和多纤毛细胞（MCCs）。的 这些细胞类型中的每一种的比例都受到严格控制，并且对于气道功能至关重要。等疾病 哮喘和慢性阻塞性肺疾病（COPD），SC亚型，杯状细胞增生， MCC的消耗导致粘液的过度产生和不能清除病原体。虽然MCC至关重要， 对于气道功能，从BCs产生MCC的细胞和分子步骤仍然不清楚。我 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 由转录因子Mycl标记。在本建议中，我将回答四个问题，以解决 气道生物学1）Mycl是已知的早期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信号传导的扰动下，Notch信号传导是SC和MCC命运的主要调节剂。2)Mycl+中间细胞是否为 过境放大人口？我将联合收割机结合原位杂交和BrdU检测， 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 将在体外气道培养系统中利用CRISPR/Cas9敲除技术， Mycl基因在MCC分化和纤毛发生中的作用。4)BC何时承诺生产监控化学品或SC？ 我将联合收割机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。我将确定BC亚型的克隆关系，以确定 MCC规范的精确时间。我假设Mycl是最早的转录因子之一， MCC规范的调节子，并标记由Notch信号传导调节的增殖群体。 此外，我假设Mycl+中间细胞是BCs的一种亚型，注定朝向MCC， 脉这些结果将建立MCC与BC差异化的路线图，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。在此过程中，我将 接受纤毛和气道生物学，单细胞RNA测序方法学和计算 单细胞RNA测序数据集的分析。在我的导师杰里米的专业建议和指导下 Reiter，UCSF内多个核心和部门的庞大资源，以及与Chan的合作 扎克伯格Biohub，我将有能力解决长期存在的气道分化问题。通过 由UCSF和杰里米·赖特的导师提供的多个职业发展计划，我将开发这两个 科学和实验室管理技能需要运行一个成功的独立的研究计划。

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.