Project Summary A conserved mechanism for keeping complex tissues organized during growth and remodeling is to separate groups of cells using compartment boundaries, which are multicellular actin-rich structures formed between adjacent cells. Compartment boundaries are typified by aligned “cables” of highly stable cell-cell interfaces, and they were first described in insect embryos nearly 40 years ago. Since then, similar boundary structures have been identified in vertebrates between different regions of the brain, gut, limb buds, and somites. While studies indicate that the loss of boundary integrity contributes to birth defects such as cranio-fronto-nasal syndrome and cancer metastasis, efforts to characterize the molecular underpinnings of these structures have been stymied by a lack of genetic tools for specifically targeting boundary cells. It was recently reported that two cell-surface proteins––the leucine-rich repeat protein Tartan and the teneurin Ten-m––are the direct spatial cues that initiate boundary formation in the Drosophila neuroectoderm. The identification of these upstream triggers finally makes it possible to answer long-standing questions concerning the nature and function of compartment boundaries. In this proposal, we will use a variety of genetic techniques to alter the expression patterns of Tartan and Ten-m in the neuroectoderm to address three significant knowledge gaps in the field. First, to identify the changes in membrane tension and adhesion that lead to boundary formation, we will use genetic engineering techniques to disrupt compartment boundaries and visualize cytoskeletal and junctional markers in live embryos. We will also use gene-swapping techniques to alter the location of boundaries to determine how their presence affects overall tissue architecture. Second, to determine how Tartan and Ten-m interact at a molecular level to trigger boundary formation, we will perform in vivo structure-function analyses to determine how Tartan controls the localization of Ten-m and which Ten-m extracellular domains are necessary for boundary formation. Third, to characterize the effector proteins downstream of Ten-m that give cell-cell interfaces at boundaries their unique physical properties, we will perform complementary biochemical and high-resolution imaging analyses. To identify putative Ten-m interaction partners, we will compare immunoprecipitation/mass-spectrometry analyses between embryos that have been enriched or depleted for compartment boundary cells. To directly visualize the nanoscale structure of compartment boundaries, we will use expansion microscopy to physically enlarge Drosophila embryos and analyze the distribution of cytoskeletal and junctional proteins that mediate cell morphology. Successful completion of this work will greatly enhance our knowledge of how compartment boundaries are formed and function. Our findings will also serve as a paradigm for understanding how these two widely expressed and...