Fstll Is Required For Lung Maturation At Birth

Normal formation and function of the lung is essential for the transition of the fetus to an air-breathing environment at birth. In the later part of gestation, the fetal lung undergoes an important maturation process, including the terminal differentiation of functional typeⅠand typeⅡalveolar epithelial cells (AEC1 and AEC2) and the production of surfactant necessary to decrease alveolar surface tension. Failure of this process always leads to respiratory distress syndrome of newborn infants.Here we showed that Follistatin-like 1 (Fstll), a secreted, follistatin-module containing glycoprotein, is essential for lung maturation and pulmonary surfactant protein homeostasis. Fstll expressed throughout the developing lungs with the highest expression at E17.5.Fstl1-/- mice died of respiratory failure shortly after birth due to impaired lung development. Although the gross phenotype was largely normal in E18.5 Fstl1-/ lungs, with similar size, lobe numbers, and lung/body dry weight ratio in comparable with their wild type littermates, Fstl1-/- lungs was strikingly condense. Histology analysis of mutant lungs showed thickened hypercellular intersaccular septa and apparently collapsed alveoli. We further confirmed that the deletion of Fstll did not affect lung branching morphogenesis at pseudoglandular stage, but delayed saccular development was visible in all Fstl1-/- embryos from E16.5 onwards. The abnormal thickness of the saccular walls in mutant lungs may be caused by the increased number of alveolar epithelial cells due to their higher proliferation rate at pseudoglandular stage. Transmission electron microscopy revealed a marked increase in the number of immature saccular epithelial cells in E18.5 Fstl1-/- lungs. In the septa of WT lungs, squamous AEC1 cells and capillary endothelial cells constituting the blood-air barrier that facilitates efficient gas exchange were clearly visible. By contrast, the septa of Fstl1-/- lungs contained an increased number of cuboidal undifferentiated alveolar epithelial cells and fewer squamous AEC1 cells and capillary endothelial cells. Consequently, the blood-air barrier was much thicker in Fstl1-/-lungs than that in WT lungs. We also examined the expressions of T1αand Aqp5 (markers of AEC-Ⅰcells) and Sftpc (marker of AEC-Ⅱcells) using QRT-PCR and immunohistochemistry. In contrast to the increased expression level of Sftpc mRNA, the expression levels of T1αand Aqp5 mRNAs were severely attenuated in E18.5 Fstl1-/- lungs. Western blot analysis showed that deletion of Fstl1 did not significantly affect the expression of pro-SP-B and pro-SP-C from E18.5 lung tissues. However, production of mature SP-B and SP-C were strikingly decreased in Fstl1-/- lungs. Therefore, saccular dysfunction caused by reduced surfactant production of immature AEC-Ⅱcells fails to prevent Fstl1-/- lung atelectasis. Collectively, these defects likely underlie the mortality of Fstl1 homozygous mutant pups.Through microarray analysis, we identified a group of genes regulating lipid homeostasis, Cell adhesion_ECM remodeling, and Immune response affected in E18.5 Fstl1-/- lungs. Moreover, deletion of the Fstl1 gene caused decreased expression of CTGF, a growth factor that regulates lung epithelial cell proliferation and differentiation.Mechanistically, Fstl1 interacted directly with BMP4, negatively regulated BMP4/Smad1/5/8 signaling, and inhibited BMP4-induced surfactant gene expression. Reducing BMP signaling activity by Noggin or Dorsomorphin rescued pulmonary atelectasis of Fstl1 deficient mice. Therefore, we provide in vivo and in vitro evidence to demonstrate that Fstl1 modulates lung development and alveolar maturation, in part, through BMP4 signaling. promoter to control the Fstl1 gene (SPC-Fstl1 transgenic mice). Western blot and q-RT-PCR analyses showed extremely high expression level of Fstl1 in the transgenic lungs. Immunohistochemical analysis demonstrated extensive activation of Fstl1 in the lung epithelium as early as embryonic day (E) 12.5. However, lung morphogenesis and lung function were not altered. Furthermore, we cross the transgenic mice with the Fstl1+/- to get the compound mice (SPC-Fstl1;Fstl1+/-). Over-expression of Fstl1 in the respiratory epithelial cells does not alter lung structure and postnatal survival and could not rescue the atelectasis phenotype of Fstl1-/- mice.