Lack of lung inflation in neonates lacking lymphatics is not due to surfactant deficiency or the effect of large pleural effusions. (A and B) Periodic acid-Schiff (PAS) staining for levels of the surfactant precursor glycogen in the lungs of Ccbe1^−/−, Vegfr3^kd/kd, and control littermate neonates. Bars, 50 µm. Representative images are shown from 5–6 littermate control and 4–5 lymphatic-deficient neonates per genotype examined from 3–4 litters in each group. Arrows indicate glycogen-rich cells in the large airways. (C) Phospholipid content of Ccbe1^−/− and littermate control neonatal lungs. Quantitative data are represented as mean and SEM from 3 control and 5 Ccbe1^−/− animals examined from 2 independent litters (P = 0.93). (D) Transmission electron microscopy of the lungs of Ccbe1^−/− neonates reveals surfactant (S) in pulmonary alveoli (left), the presence of lamellar bodies (LB) in Type II pneumocytes (middle), and Type I alveolar pneumocytes (right). Bars, 2 µm. Images are representative of 3 animals studied for each genotype from 2 independent litters. (E) Representative images of active surfactant secretion and abundant alveolar surfactant are shown in Ccbe1^−/− lungs using electron microscopy. Bars, 1 µm. (F) H-E–stained sections of the intact chest of Vegfr3^+/+, non-cyanotic Vegfr3^kd/kd, and cyanotic Vegfr3^kd/kd neonates are shown. The schematic on the left indicates the approximate plane of frontal section for each set of images. The location of lung (L) and pleural effusion (PE) are indicated. Note the presence of a very large effusion in this non-cyanotic Vegfr3^kd/kd neonate compared with that of a cyanotic Vegfr3^kd/kd littermate. Bars, 1,000 µm. (G) LYVE1-PROX1 staining for intercostal (top) and lung parenchymal (bottom) lymphatic vessels in WT1-Cre;Ccbe1^flox/flox and littermate control animals. Arrows indicate PROX1⁺ nuclei surrounded by LYVE1⁺ cell membrane in lymphatic endothelial cells. Bars, 50 µm. (H) Non-cyanotic WT1-Cre;Ccbe1^flox/flox animals after C-section at E19.5. Representative images are shown examined from 10 littermate control and 3 WT1-Cre;Ccbe1^flox/flox embryos from 2 independent litters (G and H). Bar, 5 mm.