Project Summary/Abstract The long-term goal of this project is to develop novel approaches to treating meibomian gland dysfunction (MGD), a major cause of evaporative dry eye disease in the aging population. Meibomian glands are holocrine glands that are embedded in the tarsal plate of the both the upper and lower eyelid, and excrete lipid (meibum) onto the surface of the eye to form the lipid layer of the tear film to reduce aqueous tear evaporation. Dysfunction of the meibomian gland (MGD) identified as atrophy or altered meibum secretion is a common eyelid disorder having a widespread prevalence of 39-50% in the US population with the incidence increasing with age that is widely recognized as a major cause of evaporative dry eye disease (EDED). While patients with EDED and MGD comprise from 37% to 47% of the average Ophthalmologists and Optometrists practice, management of this disease is primarily palliative due to a lack of knowledge concerning the cellular and molecular events that cause MGD. To address this UNMET NEED we have evaluated the effects of conditionally knocking out (CKO) Pparg expression in the meibomian gland using tamoxifen induced Lrig1Cre/Ppargflox transgenic mice. Our studies show that following 10 weeks of Cre induction there is a male dominant (3/5 male to 0/35 female) ablation of acini with a shift from meibocyte differentiation to Krt6a+ ductal differentiation (See Preliminary Studies). These results support our 3D spheroid culture results concerning the plasticity of meibocyte/ductal epithelial differentiation as well as suggest that there are male/female differences in the regulation of meibocyte differentiation and renewal by stem/progenitor cells. Together, these findings suggest the following hypotheses: 1) That direct loss of Pparg signaling leads to an age and sex dependent loss of meibocyte differentiation, acinar atrophy and dry eye disease; 2) That Pparg signaling transcriptionally regulates meibocyte differentiation that when lost leads to up-regulation of ductal epithelial differentiation in progenitor meibocytes; 3) That loss of meibocyte progenitor cells recapitulates loss of Pparg leading to loss of meibocyte differentiation, acinar atrophy, and dry eye disease. To test these hypotheses, we propose the following Specific Aims: 1). Determine if loss of Pparg signaling in stem/progenitor meibocytes leads to acinar atrophy and dry eye by establishing the sex (male/female) and age (1, 3 and 6 months) effects of Ppargflox knockdown in Sox9-Cre and Lrig1-Cre mice; 2). Identify the Pparg specific transcriptional profile leading to meibocyte differentiation using scATACseq and ChIP-Seq by comparing wild type to Pparg CKO mice and identify differences related to meibocyte vs ductal differentiation as well as the effects of age and sex; and 3). Establish the effects of meibocyte stem/progenitor cell ablation by conditionally knocking out Sox9 and Lrig1 using Krt14-Cre, Sox9flox and Lrig1flox mice and evaluating th...