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Wnt7b regulates mesenchymal proliferation and vascular development in the lung

Department of Medicine and the Molecular Cardiology Research Center, University of Pennsylvania, Philadelphia, PA 19104, USA

View larger version (26K): [in a new window]   Fig. 1. Targeting strategy for generation of the Wnt7blacZ mice. (A) Schematic of Wnt7b targeting construct. (B) Southern blot of a litter of newborn mice indicating the wild-type allele (10 kb) and the mutant allele (5 kb), resulting from an EcoRI restriction enzyme digest. (C) RT-PCR analysis of mRNA from E14.5 mouse lungs from Wnt7blacZ–/– embryos. The Wnt7blacZ targeted allele and the oligonucleotide used for RT-PCR are shown. No amplifiable transcripts were obtained using oligo combinations A/C or A/E (data not shown) from Wnt7blacZ–/– embryos while wild-type (+/+) mRNA produced a robust signal. Low levels of transcript were obtained using oligos D and E with lung cDNA from Wnt7blacZ–/– embryos. (D) HEK-293 cells were transfected with either HA-tagged full-length or truncated (trunc) Wnt7b cDNAs. The truncated Wnt7b cDNA represented the complete coding region from the last three exons, which are still present in the Wnt7blacZ–/– allele. Cells are counterstained with DAPI to visualize the nucleus. Scale bar: 40 µm.

View larger version (101K): [in a new window]   Fig. 2. The Wnt7blacZ allele recapitulates endogenous Wnt7b expression. lacZ staining of E12.5 (A-J,L,N,O) and E14.5 (K,M) embryos reveals that the Wnt7blacZ allele drives expression of the lacZ gene in a pattern identical to the endogenous mouse Wnt7b gene. ß-galactosidase expression is observed in the future cerebral cortex (B, yellow arrowhead), in a narrow band across the roof of the midbrain (B, black arrowhead) and the medulla oblongata (C, arrowheads). Histological sections of E12.5 embryos reveals expression in the ependymal (D,L, arrow) and the outer marginal layers (D,L, arrowhead) of the neural tube and the corpus striatum (cs), pons (pons) and the medulla oblongata (mo) (H,I). ß-galactosidase expression is observed in the interdigital mesenchyme of the forming limb bud (E, arrowhead) but is excluded from the apical ridge (F, arrowhead). High levels of ß-galactosidase expression are observed in the developing lung (G, lu) and trachea, and are restricted to the airway epithelium (J, arrowheads indicate lung epithelium, arrow indicates trachea; K, arrowheads). By E14.5, expression in the airway epithelium is restricted to the distal regions (K, compare white arrowheads with black arrowhead). ß-Galactosidase expression is observed in the developing skin at E14.5 (M), in the metanephric tubules of the developing kidney (N, arrowheads), and in the epithelium of the bile duct (O, arrowhead). Scale bars: 1.5 mm in G; 750 µm in H; 500 µm in I; 350 µm in J; 250 µm in K,L,O; 125 µm in N; 75 µm in M.

View larger version (99K): [in a new window]   Fig. 3. Wnt7blacZ–/– neonates die quickly after birth, because of respiratory distress, and display lung hypoplasia. Examination of neonates during birth revealed that a population of them did not turn pink but were cyanotic and gasped for air, quickly succumbing within minutes (A versus B). Examination of wild-type lungs showed that they were expanded by inhalation of air (C), which can be observed as air bubbles in the distal regions of the lung (D). The Wnt7blacZ–/– lungs were collapsed and hypoplastic, lacking visible air in the lung periphery (E). Histology of these lungs revealed the collapsed nature of the airways in Wnt7blacZ–/– mice and the overall smaller size of the lungs (F,G). Lungs from E12.5 Wnt7blacZ–/– and Wnt7blacZ+/– embryos were removed and stained for ß-galactosidase expression to reveal the extent of lung hypoplasia (H). Cross-sections from E14.5 wild-type and Wnt7blacZ–/– embryos further reveals the extent of lung hypoplasia (I,J).

View larger version (56K): [in a new window]   Fig. 4. Wnt7blacZ–/– embryos exhibit thin mesenchyme and reduced distal mesenchyme proliferation at E12.5. Wnt7blacZ–/– embryos at E12.5 and E14.5 exhibit thinner mesenchyme than do Wnt7blacZ+/– littermates, with the airway epithelium growing almost directly next to the mesothelium of the lung (A,B,E,F, arrows). Samples were stained for ß-galactosidase expression to better visualize the airway epithelium (A,B,E,F). Staining of lung tissue with a monoclonal antibody to phospho-histone H3, which detects mitotic cells, shows decreased staining in the distal mesenchyme of Wnt7blacZ–/– embryos at E12.5 (C,D, yellow arrowheads). By E14.5, no significant difference in cell proliferation is observed between Wnt7blacZ–/– and Wnt7blacZ+/– embryos in the lung (G,H). Quantification of cell proliferation shows that distal lung mesenchyme proliferation is reduced by approximately two-thirds in E12.5 Wnt7blacZ–/– embryos (I). Scale bars: 150 µm in A,B; 125 µm in C,D; 250 µm in E,F; 200 µm in G,H. dm, distal mesenchyme; de, distal epithelium.

View larger version (94K): [in a new window]   Fig. 5. Lung epithelial cell differentiation marker gene expression in Wnt7blacZ–/– embryos. Sections from E18.5 wild-type and Wnt7blacZ–/– embryos were analyzed by in situ hybridization using probes for SP-C, CC10 and aquaporin 5. The pattern and expression levels of SP-C and CC10 were unchanged in Wnt7blacZ–/–, embryos while the level of aquaporin 5 gene expression was reduced in Wnt7blacZ–/– embryos (A-F). Transmission electron microscopy was performed to analyze the morphology of distal airway epithelial cells (G,H). Wild-type lungs tissue revealed both AEC-1 (G, arrow) and AEC-2 cells while Wnt7blacZ–/– lung tissue lacked well defined AEC-1 cells but instead contained only cuboidal AEC-2 cells (H, arrowheads). Both wild-type and Wnt7blacZ–/– lung tissue show an extensive capillary network surrounding the distal airways (G,H asterisks). Scale bars: 250 µm.

View larger version (87K): [in a new window]   Fig. 6. Wnt7blacZ–/– embryos and neonates exhibit pulmonary hemorrhage due to defects in the vasculature. Analysis of Wnt7blacZ–/– embryos at E18.5 shows dilatation of large blood vessels compared with wild-type littermates (A,B, arrowheads; C,D). The blood vessel wall is also thicker in Wnt7blacZ–/– embryos (D, double-headed arrow). Injection of latex resin to form a cast of the embryonic vasculature reveals that the branched vessels in Wnt7blacZ–/– embryos are larger in diameter and the degree of reiterated branching is reduced (E,F, arrows). However, the main trunk vessel diameter is unchanged in Wnt7blacZ–/– embryos (E,F, arrowheads). At birth, Wnt7blacZ–/– neonates exhibit extensive hemorrhage in the lungs, primarily surrounding the larger blood vessels (G,H). Scale bars: 500 µm in A,B; 75 µm in C,D; 250 µm in G,H.

View larger version (97K): [in a new window]   Fig. 7. Defective smooth muscle integrity in Wnt7blacZ–/– embryos and mice. Close examination of blood vessels in wild-type (A, arrow) and Wnt7blacZ–/– P0 neonates (B,C) reveals several breaches in the vessel wall in Wnt7blacZ–/– mice. In some instances, very little of the structure of the wall is left (B, arrows), while in others a thicker vessel wall with several ruptures is observed (C, arrows). Staining of sections with an antibody against smooth muscle -actin shows robust staining surrounding blood vessels in wild-type neonates (D, arrows). Smooth muscle -actin staining shows reduced staining, suggesting degradation of smooth muscle surrounding some vessels in Wnt7blacZ–/– neonates (E, arrows), while other vessels show frank breaches in the hypertrophic vessel wall (F). Bronchial smooth muscle appears normal in both wild-type (G, arrowhead) and Wnt7blacZ–/– neonates (H, arrowhead). TUNEL staining shows an increase in TUNEL-positive cells in the smooth muscle of the blood vessel wall in Wnt7blacZ–/– neonates (J, arrowhead) but not in bronchial smooth muscle (K, arrow). This is not observed in wild-type littermates (I, arrow). PECAM staining reveals a normal endothelial network in wild-type (L) and Wnt7blacZ–/– neonates (M). Many large blood vessels showed rupture of the smooth muscle layer with herniation of the intact endothelial cell layer (N, arrow).

View larger version (28K): [in a new window]   Fig. 8. A model for the role of Wnt7b in lung development. (A) Wnt7b is expressed at the distal tips of the airway epithelium in a pattern similar to that observed with BMP4 and overlapping that of SHH. In addition, Wnt7b is expressed in an increasing gradient from the proximal-to-distal airway epithelium. FGFs are expressed in the mesenchyme and are known to regulate epithelial branching and proliferation. However, because BMP-4 and SHH expression is unchanged in Wnt7blacZ–/– embryos and Wnt7b expression is unchanged in Shh-null mice, Wnt7b regulates mesenchymal proliferation and differentiation through a unique pathway. (B) Lung vasculature is composed of both endothelium (red) and vascular smooth muscle (VSMC, blue), and develops in parallel with the airways (green). Loss of Wnt7b function results in defects in vascular smooth muscle differentiation and/or survival leading to a hypertrophic response (change from dark blue to light blue), degradation of the vessel wall and eventual rupture of the weakened vessels.