PROJECT SUMMARY An impressive suite of tools is available for studying prostatic epithelial cells; surprisingly few exist for prostatic stroma, even though it drives most of the prostate's significant biologic behaviors. Prostatic stroma initiates glandular development and releases paracrine signals that stimulate epithelial growth during benign hyperplasia and cancer. It also synthesizes extracellular matrix molecules that mediate regenerative repair and increase organ stiffness, factors recently associated with urinary dysfunction. Pinpointing prostatic stromal progenitors and factors involved in their proliferation and differentiation are the first steps in identifying cellular mechanisms responsible for stroma-based disease processes. Our first aim uses a mouse genetic approach to identify progenitors giving rise to prostatic fibroblasts, smooth muscle cells, perivascular cells and urinary striated muscle sphincter myocytes. We will use immunostaining and multispectral imaging to assign lineage labeled cells to two- and three—dimensional maps of prostatic stromal sub-compartments. These data will be presented using an innovative online tool that connects researchers with validated cre-expressing mouse lines and stromal- cell selective antibodies. Our second aim is to create a developing human prostatic stroma RNA and protein expression atlas to stimulate hypotheses that are relevant to prostatic disease causing behaviors of stromal progenitors identified in Aim 1. We will map expression patterns for key growth factors, receptors, and transcription factors that potentially mediate prostatic stromal and epithelial differentiation. This work complements the high-resolution developing mouse prostatic atlas we made previously for GUDMAP. Our results will be used to generate online tutorials of prostate development and we will identify homologous compartments between mouse and human. We will annotate human data when possible with the same terms used previously to annotate mouse data, providing a searchable online dataset which enables investigators to compare RNA/protein expression between mouse and human and identify conserved pathways across species. Our data will equip the prostate research community with the necessary tools to design and test new hypotheses about the pathogenic role of prostatic stroma in benign hyperplasia, inflammation, pain, and cancer.