Timed pregnancies were set up for collection of E11.5–E17.5 embryos. Embryos were fixed, dehydrated, and embedded in paraffin prior to sectioning. In older embryos, a median sternotomy was performed under microscopic guidance, and diaphragm, lungs, and heart were examined. The lungs and tracheobronchial tree were removed and weighed. Whole diaphragms were isolated from fixed thoracic tissue from E15.5 and E17.5 embryos. For lung explant culture, lungs were dissected from fresh embryos at E11.5 and E12.5 and placed on porous 24-mm (0.4-μ) polyester membranes floated in wells containing 2 ml of Dulbecco's modified Eagle's medium, nutrient mixture F-12 (11039–021, Gibco, San Diego, California, United States), supplemented with 10% fetal bovine serum, 0.3 mg/ml L-glutamine, 100 units/ml penicillin, 100 mcg/ml streptomycin, and 0.25 mcg/ml amphotericin B. Lung explants were cultured at 37 °C in 95% air/5% CO2 for up to 5 d. They were photographed daily with a dissecting microscope (MZ12.5, Leica, Wetzlar, Germany) equipped with a Leica DC500 digital camera.