PROJECT SUMMARY/ABSTRACT Many epithelial tissues are characterized by collections of cells with specific shapes and functions that are derived from a common lineage. How this diversity is introduced into the tissue through the actin cytoskeleton, which supports the creation of different cell-specific shapes, is not yet well understood. To address this question, here we will examine cytoskeletal regulation in the developing Drosophila pupal eye which has four epithelial cell types that each acquire distinctive shapes and that we have recently found become characterized by different F-actin structures, making it the ideal model. To elucidate how these cytoskeletal structures arise and their specific role in distinct cell architectures, we will use genetic, live-imaging and immunofluorescence. Our approach will establish which actin-regulatory genes are active in their formation and whether these cytoskeletal structures are shaped by local position-dependent mechanical forces. Epithelial cells are linked to each other via adhesive junctions and, especially as epithelia mature, these junctions are stabilized via connections to the actin cytoskeleton. However, we recently discovered that in the fly pupal eye, F-actin is unexpectedly removed from specific junctions providing the opportunity for us to decipher mechanisms that orchestrate the reversal of junction stability. Our genetic dissection of this process will provide insight into how actin is similarly removed from junctions in other tissues and disease states such as epithelial-to-mesenchymal transition. Erosion of F-actin, as well as the earlier generation of actin structures in the eye, likely occurs through genetic regulation, and so we will use single-nuclei RNA-seq to systematically map gene expression in individual cell types of the Drosophila eye. Our work will generate a comprehensive and dynamic understanding of gene expression crucial for morphogenesis of the cytoskeleton and its regulation in a complex epithelial tissue. Here, we will also characterize gene expression related to other aspects of eye development and maturity, revealing new regulators of tissue morphogenesis that will warrant future investigation. Uncovering the genetic bases of development, and homeostasis, will also help accelerate future development of therapeutic strategies to combat disease.