Abrogation of mesenchyme-specific TGF-β signaling results in lung malformation with prenatal pulmonary cysts in mice

Abstract

The TGF-β signaling pathway plays a pivotal role in controlling organogenesis during fetal development. Although the role of TGF-β signaling in promoting lung alveolar epithelial growth has been determined, mesenchymal TGF-β signaling in regulating lung development has not been studied in vivo due to a lack of genetic tools for specifically manipulating gene expression in lung mesenchymal cells. Therefore, the integral roles of TGF-β signaling in regulating lung development and congenital lung diseases are not completely understood. Using a Tbx4 lung enhancer-driven Tet-On inducible Cre transgenic mouse system, we have developed a mouse model in which lung mesenchyme-specific deletion of TGF-β receptor 2 gene (Tgfbr2) is achieved. Reduced airway branching accompanied by defective airway smooth muscle growth and later peripheral cystic lesions occurred when lung mesenchymal Tgfbr2 was deleted from embryonic day 13.5 to 15.5, resulting in postnatal death due to respiratory insufficiency. Although cell proliferation in both lung epithelium and mesenchyme was reduced, epithelial differentiation was not significantly affected. Tgfbr2 downstream Smad-independent ERK1/2 may mediate these mesenchymal effects of TGF-β signaling through the GSK3β-β-catenin-Wnt canonical pathway in fetal mouse lung. Our study suggests that Tgfbr2-mediated TGF-β signaling in prenatal lung mesenchyme is essential for lung development and maturation, and defective TGF-β signaling in lung mesenchyme may be related to abnormal airway branching morphogenesis and congenital airway cystic lesions.

Keywords: TGF-β signaling; airway smooth muscle cells; congenital airway cysts; lung development; lung mesenchyme.

Figure 1.

Abnormal lung development in mesenchyme-specific transforming growth factor-β (TGF-β) receptor 2 gene (Tgfbr2) conditional knockout (CKO) mice. A: body sizes of newborn Tgfbr2 CKO and wild-type (WT) mice are comparable. Bluish discoloration was apparent in Tgfbr2 CKO mice. B: lungs isolated from Tgfbr2 CKO and WT fetuses and neonates are compared side by side under dissecting microscope. Scale bar: 1 mm. C: whole mount anti-Cdh1 immunofluorescence staining of embryonic day (E) 14 lungs highlights airway epithelial branching structures. Different branching patterns between Tgfbr2 CKO and WT are marked by arrows. Scale bar: 0.4 mm. D: the numbers of left lung terminal branches between E14 Tgfbr2 CKO and WT lungs are compared. *P < 0.05 (n = 6, no. of mice).

Figure 2.

Histopathology of transforming growth factor-β (TGF-β) receptor 2 gene (Tgfbr2) conditional knockout (CKO) mouse lungs. A: the hematoxylin & eosin (H&E)-stained lung tissue sections at different developmental stages are compared between Tgfbr2 CKO and wild-type mice. B: mesenchymal cell densities between embryonic day (E) 15.5 Tgfbr2 CKO and wild-type (WT) lung tissue sections are compared. Cell nuclei were detected by DAPI staining and counted in nonepithelial area. *P < 0.05 (n = 6, no. of mice). C: the peripheral (saccular) air space at E18.5, presented as the percentage of entire tissue area, was compared between Tgfbr2 CKO and WT lung tissues. *P < 0.05 (n = 6, no. of mice). D: a H&E-stained cystic structure in postnatal day (P) 1 lung of the Tgfbr2 CKO mouse, which is lined with different epithelial cells ranging from cuboidal (arrow) to flat shapes (arrowhead).

Figure 3.

Abnormal lung morphology in embryonic day (E) 18.5 transforming growth factor-β (TGF-β) receptor 2 gene (Tgfbr2) conditional knockout (CKO) lungs that were induced from different developmental stages. Doxycycline induction was given at different time windows as indicated. Alterations of lung morphology are shown by their gross view (A) and their histopathology (B). Arrows point to the cystic lesions. WT, wild type.

Figure 4.

Blockade of transforming growth factor-β (TGF-β) receptor 2 (Tgfbr2)-mediated signaling in fetal lung mesenchymal cells resulted in altered cell proliferation and differentiation of fetal lungs. 5′-Ethynyl-2′-deoxyuridine (EdU) labeling of proliferating cells by their active DNA synthesis was detected by Alexa Fluor azide staining (red) of the lung tissue sections (A) and compared between Tgfbr2 conditional knockout (CKO) and wild-type controls (B), *P < 0.05. Epithelial cells were marked by Cdh1 immunostaining (green) and cell nuclei are counterstained with DAPI (blue). C and D: transcriptomic changes between embryonic day (E) 15.5 Tgfbr2 CKO lungs and WT controls were measured by RNA-seq. The raw data were deposited to NCBI GEO repository with the accession number GSE98138. C: gene network analysis of statistically significant genes (log₂FC ≥ 1, P < 0.05) was performed using QIAGEN Ingenuity Pathway Analysis, and the information was processed using Cytoscape 3.7.1. The nodes were colored based on their P values. *Myocd gene. D: significant changes of a group of muscle cell-associated genes (red dots) and Klf4 (green dot, a transcriptional repressor for smooth muscle differentiation) were identified. E: real-time PCR to validate the changes of three smooth muscle-related genes. *P < 0.05. F: three-dimensional (3-D) airway smooth muscle structures of E14.5 left lungs with indicated genotypes were visualized by Acta2 whole mount immunostaining (red). The airway epithelia were counterstained with Cdh1 (green). G: smooth muscle cells in E15.5 lung tissue sections were also detected by Acta2 immunostaining (green), and pulmonary vasculatures were identified by their endothelial Pecam1 staining (red, arrows). H: differentiation of airway epithelial cells (including Scgb1a1-positive Club cells and Tubb4a-positive ciliated cells) and peripheral epithelial cells (including Pdpn-positive type 1 and Sftpc-positive type 2 alveolar epithelial cells) were detected in E18.5 lungs. Cell nuclei in G and H were counterstained with DAPI (blue).

Figure 5.

Alterations of growth factor signaling in transforming growth factor-β (TGF-β) receptor 2 gene (Tgfbr2) conditional knockout (CKO) lung tissues and cells. A: embryonic day (E) 18.5 wild-type (WT) and Tgfbr2 CKO lung tissue lysates were analyzed by immunoblot. GAPDH and α-tubulin were used as loading controls. B: active β-catenin (green) in lung tissues, as detected by immunofluorescence staining, was compared between E18.5 WT and Tgfbr2 CKO mice. Cell nuclei were counterstained with DAPI (blue). C: altered signaling activities in cultured fetal lung mesenchymal cells, which were isolated from WT and Tgfbr2 CKO lungs, were measured by immunoblot. The cells were cultured with the medium containing 20% FBS and treated with additional transforming growth factor-β1 (TGF-β1, 5 ng/mL) or vehicle control for 1 h prior to sample collection. Representative data of three independent experiments are shown.

Figure 6.

ERK1/2 and GSKβ regulate Wnt canonical signaling in fetal lung mesenchymal cells. A: alterations of signal activation in primary fetal lung mesenchymal cells that were treated with ERK1/2 inhibitor U0126 (20 µM). Total cell lysates were analyzed by Western blot. α-Tubulin was used as a loading control. B: the relative intensities of the pERK1/2, pGSK3β(S9), and active β-catenin of the immunoblots in A were quantified using ImageJ and normalized with the total ERK1/2, GSK3β, and total β-catenin. C: Alterations of signal activation in primary fetal lung mesenchymal cells that were treated with GSK3β inhibitor CHIR99021 (20 μM). Total cell lysates were immunoblotted with antibodies as indicated. D: The relative intensities of the pERK1/2, pGSK3β(Y216), and active β-catenin of the immunoblots in C were quantified as described above. Compared with the untreated cells, changes of the band intensities above are all significant (P < 0.05, n = 3, no. of mice).