PROJECT SUMMARY Hearing impairment is a growing healthcare issue, which can have a large impact on an individual’s quality of life. The cochlea is made up of a large heterogenous group of cells, all working in harmony to convert mechanical stimuli into electrical signals resulting in the perception of sound. One such cell type is the otic mesenchyme cells, the most numerous cell type within the cochlea, which are essential for the maturation of normal hearing in both human and mouse. Indeed, mutations in Pou3f4, a mesenchymal specific transcription factor, causes a variety of defects in the developing cochlea, including complete loss of endocochlear potential, shortening of the cochlear duct, and decreased survival and fasciculation of spiral ganglion neurons (SGNs). We have recently discovered, using scRNA-seq, that otic mesenchyme cells are not a homogenous population, but can be separated into 4 genetically unique subpopulations. We have also shown, using immunohistochemistry with antibodies for CAR3 and TGFBI, that two of the four mesenchyme subpopulations are spatially distinct within the cochlea, revealing Car3-postive otic mesenchyme cells are located within the lateral wall, surrounding the stria vascularis, and Tgfbi-positive otic mesenchyme cells make up the spiral limbus, which is essential for SGN pathfinding. Therefore, we hypothesize that the cochlear mesenchyme cells consist of several subpopulations that are (a) spatially distinct and (b) regulate shared and unique downstream target genes. In order to test this hypothesis, we have designed two aims: (1) Determine how the spatial distribution of the cochlear mesenchyme subpopulations are changed in the absence of Pou3f4 and (2) Define the regional gene regulatory networks downstream of Pou3f4 in otic mesenchyme cells. Using a well-established in vitro mouse model and a three- pronged single-cell multi-omics approach, we will elucidate regulatory elements in each mesenchymal subpopulation, leading to the discovery of not only genes necessary for otic mesenchyme maturation, but novel genes essential for the development of key cochlear processes such as endocochlear potential. This proposal is a steppingstone in developing a more complete understanding of cochlear maturation, and specifically understanding the role of an understudied cell type crucial for normal hearing.