PROJECT ABSTRACT The lung buds undergo a process known as branching morphogenesis, which establishes the complex, arborized network of the lung1–7. During branching, a specialized population of lung progenitors at the tips of the branches, bud tip progenitors (BTPs), give rise to all epithelial cell types of the lung including cells of the proximal airway (trachea, bronchi) and cells of the distal lung (alveoli)1,8–16. We recently performed single-cell RNA sequencing (scRNAseq) on the human fetal lung from 10 to 21 weeks gestation16, and identified a novel epithelial cell population characterized by a unique gene expression profile: SCGB3A2HI/SFTPB HI/CFTRHI which we refer to as Fetal Airway Secretory (FAS) cells. We have shown that human BTPs grown in culture can give rise to FAS cells, and they have been verified in the small airways in situ. My preliminary data suggest that FAS cells are transient, dwindling in number across development. Further, our data suggests that FAS cells are uniquely human, and do not exist in mice. Since nothing is known about this newly identified population, the goal of this proposal is to interrogate the function, differentiation potential, and regulation of FAS cells. My preliminary data suggests that: (a) FAS cells may be a developmental precursor state that exists while BTPs are en route to becoming proximal airway and (b) FAS cell development is regulated by NOTCH signaling, a critical regulator of cell fate decision making in the developing lung17,18. Based on my preliminary data, I hypothesize that FAS cells are a transitional progenitor population that give rise to all airway cell types and NOTCH signaling regulates the transition from BTP-to-FAS to committed airway cell types. Using a novel cell barcoding lineage tracing approach in a human lung organoid model, I aim to 1) test the function of FAS cells, specifically if they differentiate into proximal airway cells. I also aim to 2) investigate the role of NOTCH signaling in FAS cells using gain-of-function and loss-of-function lentiviral constructs, NOTCH blocking antibodies, and pharmacologic inhibitors that activate or inhibit NOTCH signaling in an organoid model.