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.
