ABSTRACT Mucous metaplasia is commonly associated with morbidity and mortality in multiple lung diseases including fibrosis, COPD and asthma. However, the cellular and molecular mechanisms leading to mucous metaplasia in these diseases remain largely unknown. Recent lineage tracing data suggest that club cells are the cell of origin for metaplastic mucous epithelium. However, the club cell is a heterogenous population, and it remains unknown which club cell subpopulation(s) contribute to mucous metaplasia. Moreover, the molecular mechanisms leading to mucous cell differentiation are largely undetermined. We aim to address these outstanding issues in this proposal. Our single-cell RNA sequencing analysis identified three club cell subpopulations, two of which are characterized by the expression of VEGF receptor 2 (also known as Flk1 or Kdr). Significantly, deletion of Kdr leads to mucous metaplasia of the intrapulmonary airway epithelium at the early postnatal stage. Furthermore, transiently increased Kdr is required for blocking mucous metaplasia during airway regeneration following naphthalene challenge. Loss of epithelial Kdr or a hypomorphic mutation for the ligand Vegfa leads to abundant mucous cells expressing Sox9 which has been shown to regulate mucous cell differentiation in the intestine. Importantly, mucous metaplasia is also associated with reduced Kdr expression accompanied by increased SOX9 protein levels in ovalbumin (OVA)-induced asthmatic lungs. We therefore hypothesize that Vegf/Kdr signaling is a gatekeeper blocking mucous differentiation of club cell subpopulations during airway regeneration, and that suppressed Kdr promotes mucous metaplasia via Sox9 during asthma pathogenesis. We formulate three specific aims to test the hypothesis. Aim 1: To test the hypothesis that epithelial Kdr blocks club cell differentiation into mucous cells via Erk signaling. Aim 2: To test the hypothesis that Vegfa/Kdr signaling blocks mucous metaplasia of club cell subpopulations. Aim 3: To test the hypothesis that Vegfa/Kdr signaling blocks mucous metaplasia via inhibition of Sox9. Findings from these studies will provide critical insights into the cellular and molecular mechanisms that govern normal mucous cell differentiation and how the mechanism goes awry, leading to mucous metaplasia. Our study will also provide potential therapeutic targets for this common pathological entity.