Control of Epithelial Morphology and Bioenergetics by Toll Receptors During Dynamic Tissue Remodeling

Epithelial cells undergo significant changes in shape and relative position during development to build proper tissue architecture. Errors in epithelial remodeling directly contribute to some of the most common types of congenital abnormalities––neural tube defects––which affect approximately 1 in 2000 births. However, the upstream signals that control epithelial morphology 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:

1. Use newly developed CRISPR/Cas9-derived genetic backgrounds and antibodies to characterize how Toll receptors control cell polarity to trigger intercalation;
2. Apply non-destructive techniques to characterize the bioenergetics of epithelial reorganization in intact living embryos;
3. Investigate unaddressed links between Toll receptor, Rho, and G protein-coupled receptor signaling.

Our first hypothesisis 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 specific changes in mitochondrial architecture to fuel cytoskeletal and junctional reorganization. To test this, we will use the Agilent Seahorse flux analyzer to measure oxygen consumption rates and multiphoton microscopy to visualize 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 to trigger cell intercalation specifically 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, mitochondrial dynamics during epithelial remodeling.

Successful completion of these experiments will give us an understanding of how Toll receptors function at a molecular level to control cell morphology and bioenergetics during dynamic tissue remodeling, which could shed light on the cell biological underpinning of neural tube defects and other epithelia-based diseases.