studying-wnt-signaling-during-patterning-of-conducting-airways

Studying Wnt Signaling During Patterning of Conducting Airways

Wnt signaling pathways play critical roles during development of the respiratory tract. Defining precise mechanisms of differentiation and morphogenesis ...

Cincinnati Children’s Hospital Medical Center

Wntless (Wls), a gene highly conserved across the animal kingdom, encodes for a transmembrane protein that mediates Wnt ligand secretion. Wls is expressed in developing lung, wherein Wnt signaling is necessary for pulmonary morphogenesis. We hypothesize that Wls plays a critical role in modulating Wnt signaling during lung development and therefore affects processes critical for pulmonary morphogenesis. We generated conditional Wls mutant mice utilizing Shh-Cre and Dermo1-Cre mice to delete Wls in the embryonic respiratory epithelium and mesenchyme, respectively. Epithelial deletion of Wls disrupted lung branching morphogenesis, peripheral lung development and pulmonary endothelial differentiation. Epithelial Wls mutant mice died at birth due to respiratory failure caused by lung hypoplasia and pulmonary hemorrhage. In the lungs of these mice, VEGF and Tie2-angiopoietin signaling pathways, which mediate vascular development, were downregulated from early stages of development. In contrast, deletion of Wls in mesenchymal cells of the developing lung did not alter branching morphogenesis or early mesenchymal differentiation. In vitro assays support the concept that Wls acts in part via Wnt5a to regulate pulmonary vascular development. We conclude that epithelial Wls modulates Wnt ligand activities critical for pulmonary vascular differentiation and peripheral lung morphogenesis. These studies provide a new framework for understanding the molecular mechanisms underlying normal pulmonary vasculature formation and the dysmorphic pulmonary vasculature development associated with congenital lung disease.

Wntless is required for peripheral lung differentiation and pulmonary vascular development.

Midline defects account for approximately 5% of congenital abnormalities observed at birth. However, the molecular mechanisms underlying the formation of the ventral body wall are not well understood. Recent studies linked mutations in Porcupine-an O-acetyl transferase mediating Wnt ligand acylation-with defects in the thoracic body wall. We hypothesized that anomalous Wnt signaling is involved in the pathogenesis of defective closure of the thoracic body wall. We generated a mouse model wherein Wntless (Wls), which encodes a cargo receptor mediating secretion of Wnt ligands, was conditionally deleted from the developing mesenchyme using Dermo1Cre mice. Wls(f/f);Dermo1(Cre/+) embryos died during mid-gestation. At E13.5, skeletal defects were observed in the forelimbs, jaw, and rib cage. At E14.5, midline defects in the thoracic body wall began to emerge: the sternum failed to fuse and the heart protruded through the body wall at the midline (ectopia cordis). To determine the molecular mechanism underlying the phenotype observed in Wls(f/f);Dermo1(Cre/+) embryos, we tested whether Wnt/β-catenin signaling was operative in developing the embryonic ventral body wall using Axin2(LacZ) and BatGal reporter mice. While Wnt/β-catenin signaling activity was observed at the midline of the ventral body wall before sternal fusion, this pattern of activity was altered and scattered throughout the body wall after mesenchymal deletion of Wls. Mesenchymal cell migration was disrupted in Wls(f/f);Dermo1(Cre/+) thoracic body wall partially due to anomalous β-catenin independent Wnt signaling as determined by in vitro assays. Deletion of Lrp5 and Lrp6 receptors, which mediate Wnt/β-catenin signaling in the mesenchyme, partially recapitulated the phenotype observed in the chest midline of Wls(f/f);Dermo1(Cre/+) embryos supporting a role for Wnt/β-catenin signaling activity in the normal formation of the ventral body wall mesenchyme. We conclude that Wls-mediated secretion of Wnt ligands from the developing ventral body wall mesenchyme plays a critical role in fusion of the sternum and closure of the secondary body wall. Thus, impaired Wls activity in the ventral body wall mesenchyme is a mechanism underlying ectopia cordis and unfused sternum.

Mesenchymal Wnt signaling promotes formation of sternum and thoracic body wall.

Tracheobronchomalacia is a common congenital defect in which the walls of the trachea and bronchi lack of adequate cartilage required for support of the airways. Deletion of Wls, a cargo receptor mediating Wnt ligand secretion, in the embryonic endoderm using ShhCre mice inhibited formation of tracheal-bronchial cartilaginous rings. The normal dorsal-ventral patterning of tracheal mesenchyme was lost. Smooth muscle cells, identified by Acta2 staining, were aberrantly located in ventral mesenchyme of the trachea, normally the region of Sox9 expression in cartilage progenitors. Wnt/β-catenin activity, indicated by Axin2 LacZ reporter, was decreased in tracheal mesenchyme of Wls(f/f);Shh(Cre/+) embryos. Proliferation of chondroblasts was decreased and reciprocally, proliferation of smooth muscle cells was increased in Wls(f/f);Shh(Cre/+) tracheal tissue. Expression of Tbx4, Tbx5, Msx1 and Msx2, known to mediate cartilage and muscle patterning, were decreased in tracheal mesenchyme of Wls(f/f);Shh(Cre/+) embryos. Ex vivo studies demonstrated that Wnt7b and Wnt5a, expressed by the epithelium of developing trachea, and active Wnt/β-catenin signaling are required for tracheal chondrogenesis before formation of mesenchymal condensations. In conclusion, Wnt ligands produced by the tracheal epithelium pattern the tracheal mesenchyme via modulation of gene expression and cell proliferation required for proper tracheal cartilage and smooth muscle differentiation.

John Snowball

Neonatology and Pulmonary Biology-Perinatal Institute

Cincinnati Children’s Hospital Medical Center

Studying Wnt Signaling During Patterning of Conducting Airways