Project Summary During development, epithelial cells undergo programmed changes in morphology and position to create complex tissues. Studies in model organisms have identified a conserved set of effector proteins that directly alter cell shape, although the upstream pathways that coordinate these processes across large groups of cells remain poorly understood. A paradigm for studying epithelial remodeling is cell intercalation in the Drosophila neurectoderm, and it was shown that three members of the highly conserved Toll receptor family are expressed in overlapping striped patterns to organize rapid cell rearrangements in this tissue. Toll receptors are widely expressed throughout human epithelia, and they have been extensively studied in the context of innate immune signaling. However, the control of cell morphology by Toll receptors has received very little attention. The focus of this proposal is to understand how non-uniform Toll receptor expression affects cortical tension, cell-cell adhesion, and mitochondrial dynamics to control cell shape and behavior during epithelial remodeling. We will use newly developed CRISPR/Cas9-derived genetic backgrounds and antibodies to characterize how Toll receptors control cell polarity to trigger intercalation; we will apply non-destructive techniques to characterize the bioenergetics of epithelial reorganization in intact living embryos; and we will investigate unaddressed links between Toll receptor, Rho, and G protein-coupled receptor signaling. Our first hypothesis is that neighboring cells sense differences in the expression of individual Toll receptor types to increase cortical tension and decrease cell-cell adhesion. We have developed a genetic system for expressing individual receptors in a single stripe that we will use to systematically characterize and compare the effects of each Toll receptor type on cell morphology and to identify the protein domains necessary for modulating cell shape. Our second hypothesis is that rapid cellular rearrangements during neurectoderm elongation require changes in mitochondrial signaling to drive cytoskeletal and junctional reorganization. To test this, we will use multiphoton microscopy to visualize the endogenous autofluorescence of metabolic cofactors to quantify cellular redox state in live embryos during epithelial remodeling, and then use gain- and loss-of-function techniques to determine what role mitochondrial fusion and fission play in epithelial reorganization. Our third hypothesis is that Toll receptor and GPCR signaling converge to activate Rho Kinase and trigger cell intercalation in the neurectoderm. We will use gain- and loss- of-functional analyses to determine how these two signaling pathways intersect to control cortical tension, cell- cell adhesion, and mitochondrial dynamics during epithelial remodeling. Successful completion of these experiments will give us a more comprehensive understanding of how Toll receptors function at a molecular level to control cellular biomechanics and bioenergetics during dynamic tissue remodeling.

Project Narrative Defects in epithelial morphology contribute to a wide range of human pathologies, including birth defects, cancer, impaired wound healing, and inflammation, so therefore, a primary focus of developmental biology has been to uncover the mechanisms that control epithelial cell shape. Studies of epithelial tissue elongation and wound healing have shown that Toll family receptors and mitochondria are critical players in the regulation of tension and adhesion at the cell membrane, although the molecular links between these processes are still being uncovered. This proposal will address how signaling by Toll receptors and mitochondrial networks interact to drive cell shape change during tissue elongation––revealing how biomechanics and bioenergetics are coordinately regulated during embryonic development and shedding light on how these processes are misregulated in disease states.