Molecular regulation of fluid pressure homeostasis in the inner ear Project Summary A significant portion of hearing and balance disorders are caused by the unregulated accumulation of endolymph fluid and pressure within the inner ear. A key challenge in treating these diseases of elevated endolymph pressure is identifying new strategies to regain pressure homeostasis. We previously discovered a tissue-scale pressure relief valve in the epithelial tissue of the endolymphatic sac whose behavior is consistent with long observed physiologies that lacked explanations. Despite its importance in maintaining an internal environment within the ear, the molecular and cellular mechanisms by which the endolymphatic sac forms and functions remain unclear. The long-term goal of this research is to define how molecular signals during development and during homeostasis control the pressure relief valve's setpoint. Using a combination of advanced live imaging, genetics, and cell and molecular biological technologies like genome editing, we study these systemic processes in zebrafish embryos and larvae whose inner ears are optically accessible in the living animal instead of buried within the temporal bone as in mice and humans. Our prior generation of a single-cell gene expression atlas of the zebrafish inner ear identified molecular leads of signaling pathways in the endolymphatic sac. Synthesis with past work motivates our central hypothesis which posits that cells within the endolymphatic duct maintain a strong adhesive interaction, while adhesion strength at distinct subsets of cell-cell interfaces in the endolymphatic sac is reduced by adhesion protein turnover. This regulatory mechanism allows these interfaces to temporarily separate, facilitating the release of excessive pressure. We will pursue how the integration of molecular signals regulates these local cell behaviors to determine the pressure setpoint within the entire inner ear. First, we will determine how regulation of spatiotemporal differences in Wnt signaling regulate turnover rates of cell-cell adhesion complexes in subregions of the endolymphatic duct and sac. Second, we will determine the molecular responses in the endolymphatic sac that are regulated by vasopressin and how these responses integrate into physiological circuits. Third, we will determine how mechanical and calcium signals contribute to tissue contractions in the endolymphatic sac to regulate resistance to stretch. These studies will uncover regulatory mechanisms that determine the inner ear's pressure setpoint that are essential for our ability to sense sound for hearing and body acceleration for balance.