Prenatal lung epithelial cell-specific abrogation of Alk3-bone morphogenetic protein signaling causes neonatal respiratory distress by disrupting distal airway formation

Abstract

Bone morphogenetic proteins (BMPs) play important roles in regulating lung development and function although the endogenous regulatory effects of BMP signaling are still controversial. We found that BMP type I receptor Alk3 is expressed predominantly in airway epithelial cells during development. The function of Alk3 in lung development was determined using an inducible knockout mouse model by crossing epithelial cell-specific Cre transgenic mice SPC-rtTA/TetO-Cre and floxed-Alk3 mice. Abrogation of Alk3 in mouse lung epithelia from either early lung organogenesis or late gestation resulted in similar neonatal respiratory distress phenotypes accompanied by collapsed lungs. Early-induction of Alk3 knockout in lung epithelial cells caused retardation of early lung branching morphogenesis, reduced cell proliferation, and differentiation. However, late gestation induction of the knockout caused changes in cell proliferation and survival, as shown by altered cell biology, reduced expression of peripheral epithelial markers (Clara cell-specific protein, surfactant protein C, and aquaporin-5), and lack of surfactant secretion. Furthermore, canonical Wnt signaling was perturbed, possibly through reduced Wnt inhibitory factor-1 expression in Alk3-knockout lungs. Therefore, our data suggest that deficiency of appropriate BMP signaling in lung epithelial cells results in prenatal lung malformation, neonatal atelectasis, and respiratory failure.

Figure 1

Alk3 expression and conditional knockout in developing mouse lung. Alk3 protein expression was detected by immunofluorescence staining (A–D) in normal mouse embryonic lung at different developing stages, including E12.5 (A), E14.5 (B), and E18.5 (C). SPC-rtTA/TetO-Cre-induced floxed Alk3 conditional knockout was also confirmed by Alk3 immunofluorescence protein staining (D) and by RT-PCR for floxed Alk3 exon 2 (E). GAPDH RT-PCR was used as a control.

Figure 2

Phenotypes of mouse lung epithelial-specific Alk3 conditional knockout induced from E7.5. A: Gross view of neonatal lung at P1. Inflation with air was clearly seen in WT, but not in Alk3 CKO lungs. B: Collapsed lung structure in P1 Alk3 CKO mice was shown by H&E-stained tissue section. The cell shape of lining epithelial cells inside air sacs was changed from normal squamous/flat cells in WT to round/cuboidal cells in Alk3 CKO (inset). C: Surfactant production and secretion was barely detected in P1 Alk3 CKO lung by transmission electron microscopy, as shown by lack of lamellar bodies both inside cells and in air spaces (arrows). D–F: H&E-stained embryonic lung sections at different developmental stages. D: Less epithelial tubules with dilated lumen were observed in E14.5 lung of Alk3 CKO mice. E: At E16.5, peripheral airway epithelial cells still have columnar shape in Alk3 CKO lung versus cuboidal shape in WT control. F: At late gestation stage E18.5, less saccular formation with thickened mesenchyme was evident in Alk3 CKO lung.

Figure 3

Altered cell proliferation and apoptosis of Alk3 CKO lung induced from E7.5. A and B: Cell proliferation was measured by PCNA and Ki-67 immunostaining (brown color) for lungs at E14.5 and E18.5. C: Cell apoptosis was also examined by TUNEL assay (brown color). Only a few TUNEL-positive cells (arrows) were detected in E14.5 lungs of either WT or Alk3 CKO mice.

Figure 4

Expression of selected molecular markers of differentiated lung cells in Alk3 CKO lungs. A: Gene expression at mRNA level was quantified by real-time RT-PCR. *P < 0.05. B: Changes in peripheral differentiated epithelial cells were detected by SP-C immunostaining in either E14.5 (red color) or E18.5 (brown color) lungs. C and D: Immunostaining of CCSP and β-tubulin IV was used to detect Clara cells (red color) and proximal ciliated epithelial cells (brown color), respectively. E: Elastin fibers (red-black color) in lung tissues were also stained using Hart’s resorcin-fuchsin solution.

Figure 5

Pulmonary phenotypes and related cell and molecular changes in neonatal P1 lung of Alk3 CKO mice induced from E17.5 (E17.5-Alk3 CKO). A: Alk3 protein expression in lung epithelia was abrogated in E17.5-Alk3 CKO. B: Collapsed peripheral lung sac structure filled with amorphous material in E17.5-Alk CKO lung was shown in H&E-stained tissue section. C: Normal lung structure of Alk3 CKO mice induced after birth (P1-Alk3 CKO) was shown by H&E staining for the lung obtained at the end of postnatal alveolarization (age of postnatal day 30). D and E: Increased cell apoptosis and cell proliferation were detected by TUNEL (D) and PCNA immunostaining (E) in P1 lungs of E17.5-Alk3 CKO mice. F–H: Reduced gene expression of peripheral lung epithelial cell differentiation markers in P1 lungs of E17.5-Alk3 CKO mice was detected both at the mRNA level by real-time PCR quantification (F) and at the protein level by protein immunostaining for CCSP (G) and SP-C (H). *P < 0.05. Changes in CCSP-positive stained cells in terminal bronchioles were also shown under high magnification (G, insets).

Figure 6

Changes in other signal pathways of Alk3 CKO mouse lung. A: TGF-β, FGF, and SHH signaling activities in E18.5 Alk3 CKO mouse lung were detected by measuring protein phosphorylation, or protein levels of pathway-specific targeted genes. β-Actin is a loading control. B: Increased canonical Wnt signal pathway activity was observed in E18.5 Alk3 CKO lung, as indicated by increased pLRP6 and active β-catenin by Western blot. GAPDH is a loading control. C: Reduced WIF-1 gene expression at the mRNA level was detected in lung epithelial cell-specific Alk3 CKO lung tissues at indicated stages. *P < 0.05. D: Endogenous Smad1 protein expression and phosphorylation (activation) were detected for normal lung tissues at indicated stages.