Project Summary/Abstract The long-term goal of this project is to understand the mechanism of age-related meibomian gland dysfunction (MGD) and evaporative Dry Eye EDED. Recent published studies from our laboratory using Stimulated Raman Spectroscopy (SRS) to evaluate meibomian gland lipid quality (meibum) indicate that there is a decrease in protein content of normal meibum within individual glands as it moves progressively from the acinus to the orifice. This finding suggests that normal meibocyte differentiation involves removal of protein from meibum prior to release onto the tear film. Additionally our laboratory has shown that mice exposed to low humidity environmental stress, known to induce signs of EDED, display marked hyperproliferation of the meibomian gland with retention of protein in the meibum lipid suggesting that environmental stress induces abnormal or incomplete meibocyte differentiation. These findings support two alternative HYPOTHESES for the development of MGD:1) Environmental stress or aging leads to incomplete or abnormal meibocyte differentiation causing retention of protein in meibum, loss of lipid fluidity and inspissation. And 2) Aging and/or repeated environmental stress leads to depletion of meibocyte stem cells and meibomian gland dropout. These hypotheses emphasize two MAJOR GAPS in knowledge regarding meibomian gland function: GAP1, while the lipid composition of meibum and potential hormonal and other risk factors for MGD has been intensively studied, little is known regarding meibocyte differentiation and the cellular and molecular mechanisms that control this process. GAP2, although the turnover rate for the meibomian gland acinar cells has been studied, the presence of meibomian gland stem cells remains controversial and the effects of environmental stress, aging and sex are unknown. The following Specific Aims will test our hypotheses and explore these GAPS in knowledge: 1) Characterize using SRS the protein to lipid ratio (P/L) in expressed meibum from human subjects exhibiting signs and symptoms of EDED and correlate P/L ratio to EDED/MGD severity in a clinical study. 2) Identify the cellular and molecular mechanism of meibocyte differentiation and disintegration in vitro and determine the effects of environmental stress, aging and sex on meibocyte differentiation in vivo. 3) Identify the source of meibocyte and ductal epithelial renewal through lineage tracing and determine the effects of environmental stress, age and sex on meibomian gland ductal epithelial and meibocyte renewal using Confetti and the H2B-GFP/K5tTA mice and IT reconstruction.