Project Summary/Abstract Vocal fold (VF) mucosa is critical to vocal quality; poor healing ability and limitations of surgical repair have motivated strategies to engineer living tissue replacements. A lack of fundamental understanding of the complexity of VF mucosa's cellular and spatial heterogeneity is largely responsible for incremental progress being made in VF tissue engineering. The overall objective of this proposal, which is a necessary step towards achieving our long term goal of engineering replacements for VF mucosa, is to link single-cell RNA sequencing and spatial transcriptomics, at exceptional resolution, to characterize VF morphogenesis across time, location and cellular compartments and in response to injury. We will create large-scale single cell spatiotemporal atlases of VF mucosa. In specific aim 1 we will identify and characterize murine VF cell populations and their spatial distribution through embryonic and postnatal development. In specific aim 2 we will identify and characterize human VF cell populations and spatial distribution across the lifespan. Results will provide markers for characterization of proper tissue assembly and maintenance, which can then be applied as design parameters for tissue engineering approaches. Cross-species validation will be a powerful strategy for optimization, to prioritize genes and pathways for further investigation. Work in specific aim 3 will molecularly profile cell subsets involved in VF injury and repair in a murine mucosa injury model. We will identify subpopulations of fibroblasts and epithelial cells that respond to injury. Characterizing the relationship between specific cell subpopulations, their interactions, spatial location and pathways involved in VF injury and repair, will identify therapeutic avenues to mitigate VF scarring. The innovative use of transcriptional profiling as an anchor of our research strategy, will provide differentiation criteria and vital tissue engineering design parameters that can be used for the regeneration of new, normal VF mucosa.