PROJECT SUMMARY/ABSTRACT In the embryonic lung, the bronchial tree is sculpted by a process known as branching morphogenesis. Defects in branching can cause a variety of congenital malformations, such as pulmonary hypoplasia, which compromises respiratory function due to decreased lung growth. Several of these defects are caused by changes in the mechanical environment of the fetal lung, but it remains unclear how physical cues in the tissue microenvironment direct the formation of the bronchial tree. Dynamic, quantitative studies of branching morphogenesis are needed to unravel the regulatory role of mechanical forces during lung development. We hypothesize that airway branching is driven by a mechanical feedback mechanism, in which patterns of mechanical stress regulate the patterns of epithelial proliferation that sculpt the developing airways. To test this hypothesis, we will use novel experimental platforms that allow us to control and quantify the mechanical stresses within the embryonic airway epithelium. We recently developed a novel three-dimensional (3D) traction force microscopy (TFM) assay for cultured embryonic organ explants, as well as a microfluidic system to apply controlled fluid pressures to intact embryonic mouse lungs. These techniques will be combined with dynamic studies of cell proliferation and fibroblast growth factor (FGF) signaling, as well as a novel computational model of airway branching morphogenesis, to determine how physical cues regulate the growth and remodeling of the embryonic lung. In Aim 1, we will determine how mechanical stress regulates patterns of proliferation in isolated airway epithelial explants. Then, in Aim 2, we will use whole lung explant culture to uncover how mechanical forces interact with FGFs to direct both normal and hypoplastic branching phenotypes. Achieving these aims will help identify new therapeutic targets for diseases, such as congenital diaphragmatic hernia, where airway branching morphogenesis and lung growth are severely impaired. These results will also help guide pulmonary tissue engineers in their efforts to recapitulate aspects of embryonic development in the laboratory to construct engineered lung tissue.