Mesenchyme-specific deletion of Tgf-β1 in the embryonic lung disrupts branching morphogenesis and induces lung hypoplasia

Abstract

Proper lung development depends on the precise temporal and spatial expression of several morphogenic factors, including Fgf10, Fgf9, Shh, Bmp4, and Tgf-β. Over- or under-expression of these molecules often leads to aberrant embryonic or postnatal lung development. Herein, we deleted the Tgf-β1 gene specifically within the lung embryonic mesenchymal compartment at specific gestational stages to determine the contribution of this cytokine to lung development. Mutant embryos developed severe lung hypoplasia and died at birth due to the inability to breathe. Despite the markedly reduced lung size, proliferation and differentiation of the lung epithelium was not affected by the lack of mesenchymal expression of the Tgf-β1 gene, while apoptosis was significantly increased in the mutant lung parenchyma. Lack of mesenchymal expression of the Tgf-β1 gene was also associated with reduced lung branching morphogenesis, with accompanying inhibition of the local FGF10 signaling pathway as well as abnormal development of the vascular system. To shed light on the mechanism of lung hypoplasia, we quantified the phosphorylation of 226 proteins in the mutant E12.5 lung compared with control. We identified five proteins, Hrs, Vav2, c-Kit, the regulatory subunit of Pi3k (P85), and Fgfr1, that were over- or under-phosphorylated in the mutant lung, suggesting that they could be indispensable effectors of the TGF-β signaling program during embryonic lung development. In conclusion, we have uncovered novel roles of the mesenchyme-specific Tgf-β1 ligand in embryonic mouse lung development and generated a mouse model that may prove helpful to identify some of the key pathogenic mechanisms underlying lung hypoplasia in humans.

Fig. 1

Deletion of Tgf-β1 in the embryonic lung mesenchyme causes lung hypoplasia. a, b Ctgf immunostaining on E12.5 control and mutant lungs. The protein expression of the Ctgf gene, a downstream gene of the TGF-β signaling pathway, was reduced in the mutant lung, especially in the lung mesenchyme, compared with the control (dashed line marks the border between epithelium and mesenchyme) (n = 3). c Control P0 pups were healthy and had bright pink colored skin which indicates appropriate lung function and oxygenation of the blood. Conversely, mutant P0 pups were cyanotic, displayed agonal breathing and died soon after birth. d–i Cross section E18.5 embryos (d, e) and whole lung imaging (f, g) of E18.5 embryonic lungs showed that deletion of the Tgf-β1 gene led to severe lung hypoplasia (n > 40). On the other hand, deletion of Tgf-β2 (h, i) in the embryonic lung mesenchyme did not affect lung development (n = 3)

Fig. 2

Mesenchymal deletion of Tgf-β1 did not affect lung epithelial cell differentiation. a, b Lung parenchyma of mutant and normal E18.5 embryonic lung appeared very similar to each other. c–f Staining for AEC2 (Sftpc +), club (Cc10 +), ciliated (Tubb4b +), and AEC1 (T1-α +) cell markers revealed normal epithelial cell differentiation in the mutant Tgf-β1 CKO mouse lungs. g, h Staining for α-Sma and E-Cadherin to highlight lung smooth muscle and epithelial cells, respectively. Differentiation of smooth muscle cells (vascular and bronchial smooth muscle cells and pericytes), and spatial organization of the epithelia cells were also comparable between control and mutant embryonic lungs (n = 4)

Fig. 3

Lack of mesenchymal expression of Tgf-β1 increased cellular apoptosis. a, b Control and mutant E13.5 lungs were collected and analyzed for the presence of apoptotic cells. Lack of mesenchymal expression of Tgf-β1 was associated with markedly increased apoptosis of the lung epithelium, mesenchyme, and mesothelium (white arrowhead) (n = 3) (dashed line marks the border between epithelium and mesenchyme). c–e Meanwhile, the number of proliferating cells was similar between mutant and control E13.5 lungs (n = 3)

Fig. 4

Branching morphogenesis and FGF10 signaling pathway were inhibited in the Tgf-β1 CKO lungs. a–c To detect changes in the rate of lung branching morphogenesis, we collected E13.5 control and mutant lungs and counted the number of terminal buds in the left lung lobe. Lung branching was significantly delayed in the freshly isolated mutant lungs (n = 3). d–j Real-time qRT-PCR (d) and in situ hybridization (e–j) revealed that the FGF10 signaling pathway was strongly inhibited in the Tgf-β1 CKO embryonic lungs as suggested by the significantly reduced mRNA expression of the Bmp4, Shh and Fgf10 genes (n = 4) (**p < 0.01, *p < 0.05)

Fig. 5

Branching morphogenesis in the mutant lung is rescued in vitro. a–e Control and mutant E12.5 lungs were harvested and cultured on air–liquid interface on semipermeable polycarbonate porous membranes for 72 h. Even though at 72 h the number of terminal buds was still reduced in the mutant lung compared with the control lung, the rate (slope of the interpolated line) at which control, and mutant embryonic lungs branched in culture over time did not differ (n = 4) (*p < 0.05). f, g Whole mount Pecam1 staining on E12.5 control and mutant lungs showed that the vascular system was immature and less organized in the Tgf-β1 CKO lungs (n = 5)

Fig. 6

Tgf-β1 deletion in the lung mesenchyme interferes with the phosphorylation of several proteins in the embryonic lung. a, b Black and white photo of the scanned arrays. Colored rectangles identified five proteins whose change of phosphorylation level was clearly changed. c Average and standard deviation of the phosphorylation levels of Hrs, Fgfr1, P85, Vav2, and c-Kit (*p = 2E–06, **p = 0.009, ***p = 0.1, ^Δp = 0.036, ⁺p = 0.034). d, e Wild-type E12.5 lungs were cultured with DMSO, wortmannin (WM), PD173074 (PD) or WM+PD for 2 days to determine the effect of the inhibiton of Pi3k and Fgfr1 on lung branching morphogenesis. Both drugs inhibited lung growth and branching (n = 3) (^δp < 0.05)

Fig. 7

Deletion of Tgf-β1 at later gestational stages led to progressively weaker lung phenotypes. a–e Deletion of the Tgf-β1 gene at E11.5 and E13.5 was associated with lung hypoplasia. Meanwhile, Tgf-β1 deletion at the E15.5 stage did not produce a gross lung phenotype (n = 3). f–i E15.5 pregnant females were fed doxycycline chow until birth of the pups. Lungs were collected from 2 months old, control and mutant animals for analysis. Tgf-β1 knockout at the E15.5 stage did not affect postnatal lung growth nor its alveolarization (n = 4)