Interplay between mechanical forces and retinoic acid in lung development

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PROJECT SUMMARY Breathing after birth requires coordinated development of the airway epithelium and its surrounding mesenchyme and mesothelium. In the fetus, these tissues form in response to exogenous forces from fluid pressure within and around the developing lungs that is normally high in the lumen of the airways, thus generating a positive transpulmonary pressure. The mechanical environment of the fetal chest cavity is disrupted by conditions such as congenital diaphragmatic hernia (CDH), which reduces or reverses the pressure across the developing lungs and leads to pulmonary hypoplasia, a major cause of neonatal mortality. Although several biochemical signals, including retinoic acid (RA), have been implicated in the pathogenesis of CDH, the mechanical forces and signaling downstream of transpulmonary pressure that regulate lung development are unknown. Our preliminary and published data suggest that transpulmonary pressure itself regulates the RA-biosynthesis pathway, airway epithelial branching morphogenesis, and airway smooth muscle differentiation. Here, we propose to take advantage of our innovative microfluidic platforms, tissue- specific knockout mice, fluorescent reporter mice, and mouse models of CDH to uncover the molecular mechanisms that connect pressure, RA signaling, and morphogenesis and differentiation within the embryonic lung. We will combine these approaches with time-lapse imaging of lung explants, single-cell transcriptomic analysis, and real-time fluorescent force sensors. In Specific Aim 1, we will test the hypothesis that pressure activates the mechanosensor Yap in a tissue-specific manner to regulate the spatiotemporal pattern of expression of genes involved in the RA-biosynthesis pathway. In Specific Aim 2, we will uncover the effects of pressure and tissue-specific synthesis of RA on airway epithelial growth and morphogenesis and airway smooth muscle differentiation. In Specific Aim 3, we will measure the relative effects of pressure and RA signaling on tension, strain, and fluidity within the epithelium, mesenchyme, and mesothelium. This work will, for the first time, identify the tissue-specific mechanical forces and molecular signaling downstream of transpulmonary pressure that regulate early morphogenesis of the lung. We expect that our findings will suggest novel therapeutic targets for the treatment of defects in lung development.