The subject of this proposal is the cell biology of lung development, particularly regarding connective tissue morphogenesis. The studies described below will focus on the early morphogenic events, when the primitive lung bud epithelium (endoderm) is surrounded by splanchnic mesoderm. During this time the mesodermal condensations impose upon the epithelium a precise branching morphogenic pattern. The migratory activity of the mesenchymal cells, their interactions with extant matrix, and their secretion of new extracellular matrix (ECM) will be investigated. We hypothesize that mesenchyme cells exist in at least two functional states: the first involves cell-ECM interactions that result in migratory activity. The second state involves cell-ECM interactions that result in stationary cells exerting tractional forces on the ECM. To test this hypothesis we propose: 1) to biochemically "dissect" the lung mesenchyme by carefully controlled enzymatic digestion. 2) to prepare reconstituted 3-dimensional matrices seeded with mesenchymal cells. 3) to examine these two preparations plus intact lung mesenchyme for interations between enbryonic cells and the ECM. This will be accomplished by pairwise co-distribution studies of specific cytoskeletal and specific ECM components using double immunolabeling technology. Studies will also be conducted to provide a thorough biochemical and immunochemical characterization of ECM polypeptides in sequential stages of development from lung bud formation until neonatal stages. This will be accomplished by gel electrophoresis (native and SDS), chromatography, radioimmunoassay, enzymatic "dissection", as well as double immunofluorescence, and double immunoelectron microscopy on semi-thin and ultra-thin frozen sections. In addition; polarization, differential interference, phase and reflection contrast microscopy will be employed. It is hoped that by using a combined microscopic, immunological and biochemical approach to identify and locate specific extracellular, cell surface and cytoskeletal components much can be learned regarding the activities of early cell populations involved in normal lung morphogenesis. Understanding the architecture of normal lung primordia will provide insights into maldevelopment of these structures, and allow more critical investigation into the abnormal molecular anatomy of disease lungs.