PROJECT SUMMARY Corneal epithelium (CE) is a self-regenerating stratified squamous tissue that protects the rest of the eye by serving as the anterior-most barrier.1, 2 Our long-term goal is to identify the regulatory networks that govern the CE stratification and homeostasis, facilitating better understanding of the molecular basis for sight-threatening corneal disorders. Previously, we demonstrated that the Krüppel-like factors Klf4 and Klf5 play crucial non- redundant roles in maturation and maintenance of the ocular surface.2-17 In this proposal we seek to determine their role in regulating distinct cellular and molecular pathways of CE stratification and homeostasis, and elucidate how defects in these pathways affect CE plane of division and genetic stability, causing ocular surface diseases. We will employ complementary and innovative cell culture and imaging systems, transgenic mouse models and Next-Gen sequencing approaches to test the novel central hypothesis that `Klf4 and Klf5 orchestrate CE cell stratification and homeostasis by coordinating the CE cell plane of division and genetic stability in a TGF-, BMP6-, and Pard3-dependent manner'. This hypothesis was formulated based on our published work, 2-16 and exciting preliminary results. We will test this hypothesis by pursuing three Specific Aims. In Aim 1, we will test if KLF4 and KLF5 play key roles in regulating CE cell shape, apical-basal polarity (ABP) and plane of division, thereby elucidating the link between these transcription factors and the cellular processes that regulate CE stratification. In Aim 2, we will test if CE stratification is driven by a crosstalk between transcription factors Klf4 and Klf5, and signaling network involving TGF and BMP6 pathways, thereby elucidating the molecular mechanisms that regulate CE stratification and homeostasis. In Aim 3, we will test if Klf4 is a key regulator of genome stability that protects the CE from radiation-induced DNA damage and tumor development by upregulating the ABP gene Pard3 (also called Par3) expression. By delineating the regulatory pathways of corneal epithelial differentiation, stratification and carcinogenesis, and elucidating the molecular underpinnings of ocular surface disorders such as pterygium and ocular surface squamous neoplasia (OSSN), this proposal directly addresses the NIH mission of `seeking fundamental knowledge about the nature and behavior of living systems' and offers the potential for translation to ocular surface disorders which account for considerable healthcare burden in the world. The proposed research is significant as its anticipated outcomes will identify the molecular factors and pathways important for normal growth and function of CE cells, and their deficiencies that lead to sight-threatening corneal disorders.