Cellular and molecular regulation of airway multiciliated cell specification and differentiation

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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.