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First published online 8 February 2006
doi: 10.1242/dev.02273

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C/EBP is required for lung maturation at birth

Division of Pulmonary Biology and Neonatology, Cincinnati Children's Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, OH 45229, USA.

View larger version (61K): [in a new window]   Fig. 1. Deletion of Cebpa. Lung sections from control (A) and Cebpa/ (B) mice were prepared at E18.5 and stained using a C/EBP polyclonal antibody. Nuclear C/EBP staining was observed in alveolar epithelial type II cells of control littermates and was absent or decreased in Cebpa/ mice. Scale bar: 100 µm.

View larger version (79K): [in a new window]   Fig. 2. Pulmonary immaturity in Cebpa/ mice. (A) Lung sections prepared at E18.5 and stained with Hematoxylin and Eosin revealed normal lung inflation and histology, indicated by saccular structures lined by squamous type I cells and cuboidal type II cells, in control littermates. (B) Severe atelectasis, pulmonary congestion, reduced septation, thickened mesenchyme and columnar epithelium lining the peripheral saccules were observed in the Cebpa/ lung. Scale bar: 50 µm. (C,D) Consistent with the observed immaturity in the Cebpa/ lung, PAS staining demonstrated increased glycogen content in the bronchiolar epithelium of Cebpa/ mice (D) compared with control littermates (C). Scale bar: 1 µm. Electron microscopy was performed on lungs from control (E,F) and Cebpa/ (G,H) mice at E18.5. Squamous type I cells (black, arrowhead) and cuboidal type II cells (white triangle) containing numerous lamellar bodies (*), apical microvilli (MV) and highly organized rosette glycogen were observed in the lungs of control mice. Lamellar bodies were observed in the lumen of peripheral airspaces and secreted surfactant (thin arrow) was identified in the airspaces (E). By contrast, lamellar bodies, secreted surfactant, and type I cells were not observed in the lungs of Cebpa/ mice (G). Cytoplasmic glycogen (GLY) was dispersed and apical microvilli were smaller in epithelial cells lining the abnormal lung tubules (H). Micrographs are representative of three Cebpa/ mice and littermate controls.

View larger version (84K): [in a new window]   Fig. 3. Alveolar type I and type II cell maturation is disrupted in Cebpa/ mice. Serial sections of lung from E18.5 control and Cebpa/ mice were stained for FOXA1 (A,B), TTF1 (C,D) and Aqp5 (E,F). In Cebpa/ mice, the cuboidal epithelium of the peripheral lung saccules homogeneously expressed FOXA1 and TTF1, indicative of structural immaturity. Consistent with pulmonary immaturity, Aqp5 staining, a marker of squamous type I cells, was absent in lungs of Cebpa/ mice. Scale bar: 25 µm.

View larger version (71K): [in a new window]   Fig. 4. C/EBP regulates expression of surfactant proteins in vivo. Serial sections of lung from control and Cebpa/ mice at E18.5 were stained for mature SP-B (A,B) and proSP-C (C,D). Staining for mature SP-B and proSP-C was markedly reduced in conducting airways and peripheral lung saccules in Cebpa/ mice. Scale bar: 25 µm.

View larger version (23K): [in a new window]   Fig. 5. C/EBP regulates expression of surfactant proteins and ABCA3. Surfactant protein mRNAs and surfactant proteins were decreased in Cebpa/ mice. Total lung RNA was prepared from fetal mouse lung at E18.5. S1 nuclease assay was performed with 3 µg of total RNA for estimation of SP-A, SP-B, SP-C, SP-D and ribosomal protein L32 mRNA. (A) Protected fragments were standardized to the internal control L32 and normalized to control littermates. (B) Decreased surfactant proteins (SP-A, SP-B, SP-C and SP-D) were detected in lung homogenates from Cebpa/ mice. (C) Western blot analysis demonstrated that ABCA3 protein was reduced after deletion of Cebpa. Lung homogenates were prepared from Cebpa/ and littermate controls at E18.5. Surfactant proteins and ABCA3 were identified by western blot analysis, quantified by densitometry and normalized to controls. n=6 animals per group; *P<0.05 versus control.

View larger version (112K): [in a new window]   Fig. 6. C/EBP influences maturation of the capillary-epithelial interface. Lung sections from Cebpa/ mice and control littermates were obtained at E18.5 and immunostained for vWF and -SMA. In controls (A), an extensive vascular network was observed in which vessels are located in close apposition to squamous type I cells. In Cebpa/ mice (B), a relatively undeveloped capillary bed, with vessels embedded within the thickened mesenchyme of the immature lung saccules, was observed. Expression of -SMA was detected in smooth muscle cells surrounding the bronchioles in both control (C) and Cebpa/ (D) mice. Scale bar: 50 µm.

View larger version (62K): [in a new window]   Fig. 7. C/EBP does not regulate C/EBPß and C/EBP in vivo. C/EBP mRNA was decreased but expression of C/EBPß and C/EBP mRNA was maintained in lungs from Cebpa/ mice. Total lung RNA was prepared from fetal mouse lung at E18.5. RNAse protection assay was performed with 7 µg of total RNA for estimation of C/EBP, C/EBPß, C/EBP, and ribosomal protein L32 mRNA. Protected fragments were standardized to the internal control L32 and normalized to control littermates.

View larger version (50K): [in a new window]   Fig. 8. C/EBP regulates expression of TGFß2. (A) Control section. (B) Increased TGFß2 immunostaining was detected in lungs of Cebpa/ mice at E18.5. TGFß2 expression was detected in mesenchymal tissue, type II cells and in the epithelial lining of the developing airway along a proximodistal gradient. Scale bar: 25 µm.

View larger version (80K): [in a new window]   Fig. 9. Decreased expression of C/EBP in Titf1–/– mice and Foxa2/ mice. Lung sections from Titf1+/–, Titf1–/–, Foxa2flx/flx and Foxa2/ mice were prepared on E18.5 and immunostained for C/EBP. C/EBP was not altered in lungs from Titf1+/– (A) and Foxa2flx/flx (C) mice. Titf1–/– mice (B) have a rudimentary bronchial tree associated with absent peripheral lung formation in which C/EBP staining was markedly decreased or absent. Expression of C/EBP was decreased in respiratory epithelial cells in the lungs of Foxa2/ (deficient) mice (D). Scale bar: 25 µm.

View larger version (21K): [in a new window]   Fig. 10. C/EBP activates gene transcription in vitro. C/EBP enhanced luciferase activity in HeLa cells after co-transfection with Sftpa (A), Sftpb (B), Sftpc (C) and Abca3 (D) promoter constructs in a dose-dependent manner. *P<0.05 versus control.

View larger version (24K): [in a new window]   Fig. 11. C/EBP regulates Foxa2 in vitro and in vivo. FOXA2 mRNA levels were decreased in lungs from Cebpa/ mice (A). Total lung RNA was prepared from fetal mouse lung at E18.5 from dams that were treated with doxycycline from E0 to E18.5. RNase protection assay was used to estimate FOXA2 and ribosomal protein L32 mRNA. Protected fragments were standardized to the internal control L32 and normalized to control littermates. (B,C) HeLa (B) and H441 (C) cells were transfected with reporter plasmid pGL3-Foxa2p1.6 with increasing amounts of expression plasmid pCMV5-C/EBP. FOXA2 promoter activity was determined by relative luciferase activity and normalized for co-transfection using ß-galactosidase. Plasmid pcDNA was used as an empty control. Activation of FOXA2-luciferase construct by C/EBP was dose dependent in both HeLa and H441 cells. *P<0.05 versus control.

View larger version (16K): [in a new window]   Fig. 12. Interactive roles of Cebpa, Foxa2 and Titf1. C/EBP, FOXA2 and TTF1 reciprocally or synergistically interact in a transcriptional network to regulate genes required for maturation and function of the lung at birth.

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