Uncovering the mesenchyme-specific roles of planar cell polarity in lung development - PROJECT SUMMARY Breathing after birth requires a large alveolar epithelial surface and a thin mesenchymal barrier to promote adequate gas exchange. In the fetus, this gas-exchange surface begins to form during the saccular stage of lung development, when the distal ends of the epithelium expand into primary saccules at the same time as the surrounding mesenchyme thins. Sacculation is arrested or delayed in premature infants, thus resulting in significant decreases in lung function that persist into adulthood. Understanding the signals that promote mesenchymal thinning would therefore permit strategies to treat sacculation delays. In the embryo, signaling by noncanonical Wnts through the core planar cell polarity (PCP) pathway coordinates cellular rearrangements that lead to convergence and extension of epithelial tissues. Our preliminary and published data show that the PCP component, Vangl, is required in the lung mesenchyme but not the epithelium for sacculation. Additionally, our data show that several core PCP pathway components, including Celsr, are absent from the lung mesenchyme, suggesting that Vangl functions through a novel, mesenchyme-specific pathway to promote mesenchymal thinning. Furthermore, we find that expression of the noncanonical Wnt5a in the mesenchyme is required for sacculation. Here, we propose to take advantage of our innovative live-imaging platforms, tissue- specific knockout mice, and endogenously tagged PCP-reporter mice to uncover the molecular, cellular, and tissue-level mechanisms that connect Vangl to mesenchymal thinning during sacculation. We will combine these approaches with atomic force microscopy, real-time fluorescence-based force sensors, super-resolution imaging, unbiased proteomics, and single-cell transcriptomic analyses. In Specific Aim 1, we will determine whether Vangl regulates the active or passive mechanical properties of the sacculating mesenchyme by defining the effects of Vangl expression on cell motility and mechanical stiffness, respectively. In Specific Aim 2, we will test the hypothesis that Vangl promotes mesenchymal thinning by polarizing mesenchymal cells, and identify the molecular components of the mesenchyme-specific PCP pathway. In Specific Aim 3, we will define whether Vangl is activated by Wnt5a to promote mesenchymal thinning and extracellular matrix remodeling during sacculation. This work will, for the first time, uncover the physical properties and molecular signaling within the mesenchyme that promote late-stage lung development, and unveil the molecular players and cellular consequences of a novel, mesenchyme-specific Vangl pathway. Our findings will have broad implications for both lung developmental biology, in particular, and PCP signaling, in general.
