PROJECT SUMMARY The mechanisms involved in lung repair and regeneration are still poorly understood and currently no clinical techniques are available to specifically induce lung repair. In young tissues, the lung is appreciated to repair and regenerate in response to damage. However, lung diseases such as idiopathic pulmonary fibrosis, are largely considered diseases of aging and subsequent disordered repair. Current treatment options available are limited, and mainly provide an option to slow disease progression, but none offer the ability to repair existing damage. Improved understanding of repair and regenerative mechanisms could drastically improve the treatment options available for these terminal fibrotic conditions. Of the cells studied in repair and regeneration, mesothelial cells are largely excluded, presenting a gap in the knowledge base. While pleural mesothelial cells (PMCs) are largely quiescent in healthy conditions, our preliminary data shows recruitment and migration of PMCs in a mouse model of transient fibrotic injury. Compelling data also exists for PMC recruitment and migration in a mouse model of lung regeneration. Contradicting this, other work has suggested that PMCs serve a role to stimulate fibrotic progression, leading to confusion surrounding the function of these cells. The lack of study and contradictory evidence regarding PMC functions in lung injury, fibrosis and repair sets the stage for detailed study of this cell population and the signaling and functional roles it may play in these contexts. Using a combination of careful lineage-tracing experiments, we will investigate PMCs in the bleomycin injury model, as well as in the pneumonectomy compensatory growth and regeneration model. We will subsequently collect these cells for further genomic analysis to assess expression of genes consistent with injury progression or controlled repair. Our initial investigations into the signaling of PMCs has capitalized on the various lung single-cell RNA sequencing dataset that are publically available. In multiple human and mouse datasets, the cell clusters identified as mesothelium represent the exclusive lung cell type that expresses the Wnt mediator R-spondin 1 (Rspo1). Our preliminary data confirms PMC to be highly expressive of Rspo1. As Wnt signaling is a pathway that has been well explored in contexts of injury and repair, this proposal will explore the ability of PMCs to modulate Wnt signaling in the local environment but using organoid co-culture. The goal of these studies is to provide context for the effect of these cells on specific cell types involved in lung injury and repair. Overall, the studies may reveal a unique and unexplored potential to target pleural mesothelial cells and explore their expanded role in lung function/dysfunction.