PROJECT SUMMARY In this application entitled “KLF4 and WNT5A in esophageal epithelial differentiation and stratification,” we seek to define the molecular pathways of esophageal epithelial differentiation and stratification and the perturbations of these pathways that lead to esophageal diseases. The PI is an experienced investigator and expert in the Krüppel-like factors (KLFs), animal models of disease, and esophageal squamous cell biology, and he is supported by a superb research team, a collegial intellectual environment, and exceptional resources and facilities. The proposed research is highly significant, addressing the NIH mission of “seeking fundamental knowledge about the nature and behavior of living systems” and offering the potential for translation to esophageal disorders such as defective wound healing after injury (e.g. radiation, infection, eosinophilic esophagitis) and diseases such as esophageal squamous cell cancer (ESCC), which together account for considerable morbidity, mortality, and healthcare burden in the United States and throughout the world. In complementary approaches, we will employ novel mouse models, innovative two-dimensional and three- dimensional culture systems, and next generation sequencing approaches to test the hypothesis that KLF4 is a key regulator of esophageal epithelial differentiation and stratification and that studies of differentiation and stratification in normal epithelia provide the foundation for insights into how these processes are disrupted during diseases such as ESCC. To address this hypothesis, we will pursue the following interrelated Specific Aims: (1) We will define the pathways of normal differentiation and stratification through mechanistic studies of KLF4 and WNT5A (a) functional interactions and (b) via CDC42 and other downstream targets; and (2) We will delineate the molecular underpinnings of ESCC through functional studies of KLF4 and WNT5A in vitro and in vivo. The PI and his team have expertise in all techniques proposed in this application, and all models are currently in hand. In sum, we anticipate that these studies will provide substantial insight into the regulatory pathways of esophageal epithelial differentiation, stratification, and carcinogenesis, offering the potential for eventual translation of this knowledge to human esophageal diseases.