The pituitary is critical to all endocrine systems. In recent work, we have optimized methods to generate high quality single cell omics datasets using individual frozen mouse pituitaries. Together with the accompanying advances in single cell bioinformatic analysis platforms developed by us and our collaborators, these capabilities and results provide the foundation to achieve comprehensive insight into the layered epigenetic regulatory mechanisms underlying cell physiology and disease processes, spanning from single genes resolved in detail in each cell type to the global gene regulatory responses across the diverse cells of the pituitary. Results to date suggest an expected plasticity of cell state and cell type across pituitary cell systems during the reproductive cycle and during development of an important pathophysiological condition, primary hypothyroidism. We will study the overall hypothesis that a profound and previously unappreciated plasticity of mature pituitary cell types underlies normal physiology and disease. Because regulation of alternative gene splice site usage is important in pituitary cell type function, we will incorporate comprehensive study of the dynamics of pituitary cell type alternative splice usage, alternative promoter usage and lncRNAs into our single cell platforms using single cell long read methods. We propose to test hypotheses identified in single cell in vivo datasets for the signaling and epigenetic mechanisms regulating Fshb, a key hypothalamic-pituitary-gonadal axis regulator gene that is transcriptionally controlled (Aim 1). We will elucidate the coordination of regulatory processes across multiple pituitary endocrine cell types underlying pituitary plasticity during the reproductive cycle, using both single cell short read and long read and refined analysis methods (Aim 2). Focusing on a pituitary disease that causes transdifferentation among pituitary cell types, we will apply these single cell methods to delineate the multi-cell type mechanisms mediating pituitary adaptations to primary hypothyroidism in mouse models and investigate the underlying mechanisms for these processes (Aim 3). This research will address key questions relevant to normal physiological processes and disease and provide data and methods to advance research for both endocrine researchers and the broader community.