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. 2013 Sep;140(17):3589-94.
doi: 10.1242/dev.095471. Epub 2013 Jul 24.

Gpr177 regulates pulmonary vasculature development

Ming Jiang  1 , Wei-yao Ku, Jiang Fu, Stefan Offermanns, Wei Hsu, Jianwen Que
Affiliations

Gpr177 regulates pulmonary vasculature development

Ming Jiang et al. Development. 2013 Sep.

Erratum in

  • Development. 2013 Oct;140(19):4117

Abstract

Establishment of the functional pulmonary vasculature requires intimate interaction between the epithelium and mesenchyme. Previous genetic studies have led to inconsistent conclusions about the contribution of epithelial Wnts to pulmonary vasculature development. This discrepancy is possibly due to the functional redundancy among different Wnts. Here, we use Shh-Cre to conditionally delete Gpr177 (the mouse ortholog of Drosophila Wntless, Wls), a chaperon protein important for the sorting and secretion of Wnt proteins. Deletion of epithelial Gpr177 reduces Wnt signaling activity in both the epithelium and mesenchyme, resulting in severe hemorrhage and abnormal vasculature, accompanied by branching defects and abnormal epithelial differentiation. We then used multiple mouse models to demonstrate that Wnt/β-catenin signaling is not only required for the proliferation and differentiation of mesenchyme, but also is important for the maintenance of smooth muscle cells through the regulation of the transcription factor Kruppel-like factor 2 (Klf2). Together, our studies define a novel mechanism by which epithelial Wnts regulate the normal development and maintenance of pulmonary vasculature. These findings provide insight into the pathobiology of congenital lung diseases, such as alveolar capillary dysplasia (ACD), that have abnormal alveolar development and dysmorphic pulmonary vasculature.

Keywords: Hemorrhage; Klf2; Lung morphogenesis; Mouse; Wls; Wnt; Wntless.

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Figures

Fig. 1.
Fig. 1.
Loss of epithelial Gpr177 in the Shh-Cre;Gpr177loxp/loxp (Gpr177Δ/Δ) mutants leads to reduced Wnt signaling and severe pulmonary hemorrhage. (A,B) Deletion of epithelial Gpr177 reduces canonical Wnt signaling in both the epithelium and mesenchyme of the E12.5 lungs, as shown by the Axin2-lacZ allele. (C) Whole-mount microscopy shows reduced branching in the left lobe of E12.5 mutant lung. Arrowheads indicate lateral secondary branches in the normal lung. (D) Gpr177 deletion leads to hypoplastic lung with severe hemorrhage. (E) Hematoxylin and Eosin staining shows interstitial space in the mutant lung is filled with blood cells. ep, epithelium; me, mesenchyme; lu, lung; st, stomach. Scale bars: 50 μm.
Fig. 2.
Fig. 2.
Loss of epithelial Gpr177 leads to limited alveolar development in the E18.5 lung. (A) Proximal-distal specification of epithelial progenitor cells is unaffected in the E14.5 mutant lung. Proximal and distal epithelial progenitor cells are labeled with Sox2 and Sox9, respectively. (B) Sox2 is maintained in the proximal airways of the normal and mutant lungs at E18.5. (C,D) Clara cells (Scgb1a1+) and ciliated cells (α-acetylated tubulin+) are present in the proximal airways of the normal and mutant lungs. (E-G) The numbers of type I cells (T1α+, arrowheads) and type II cells (SpC+ and ABCA3+, arrowheads) are reduced in the mutant lungs. The insets are the high magnification view of the boxed region. Scale bars: 50 μm.
Fig. 3.
Fig. 3.
Deletion of epithelial Gpr177 disrupts mesenchymal morphogenesis. (A) The density of blood vessels (Pecam1+) is reduced in the E14.5 mutant lung. (B) Gpr177 deletion leads to reduced numbers of vascular and airway smooth muscle cells. (C) Representative section to show decreased proliferating cells (pH3+) in the mesenchyme of the mutant lung. Dotted lines indicate the basement membrane. (D) Quantification of proliferating cells in the epithelium and mesenchyme of the control and mutant lung. (E) Loss of Gpr177 results in reduced expression of the matrix protein laminin. Asterisks indicate bronchiolar airways. (F) Gpr177 deletion leads to the reduced expression of tenascin-C (TnC). (G) The expression of Klf2 is reduced in the mesenchyme of the Gpr177 mutant lung. (H) Deletion of Gpr177 leads to decreased protein levels of Klf2 in the E16.5 and E18.5 lung, as shown by western blot analysis. Scale bars: 50 μm.
Fig. 4.
Fig. 4.
Wnt/β-catenin signaling maintains vascular smooth muscle cells through Klf2. (A) Tamoxifen was injected into mothers bearing E11.5 SMMHC-CreER;β-cateninloxp/loxp mutants and their littermates, and the embryos were harvested at E14.5. (B-C′) Loss of β-catenin leads to decreased number of smooth muscle cells surrounding the blood vessels and airways in the SMMHC-CreER;β-cateninloxp/loxp mutants. There is a discontinuous smooth muscle ring surrounding the blood vessels (arrowheads) and airways (arrows). Asterisks indicate blood vessels. (D,E) β-Catenin deletion leads to reduced levels of Klf2 in the smooth muscle cells of the blood vessels (arrowheads) and airways (arrows). Klf2 expression is maintained in a subpopulation of mesenchymal cells (SMA negative). (F) Activation of the Wnt/β-catenin pathway by co-transfection with plasmids encoding active β-catenin-Lef fusion protein (CatcLef) or Wnt3a leads to increased promoter activity of the Klf2 gene in rat pulmonary vascular smooth muscle PAC1 cells. Three potential β-catenin-binding sites are present in the 3 kb promoter region of the Klf2 gene. A dual luciferase assay was used to quantify the promoter-driven luciferase activities. (G) A point mutation was generated for each of the three β-catenin-binding sites (M1, M2 and M3). Disruption of M1 and M2, but not of M3, significantly diminishes the luciferase activity driven by the Klf2 promoter. Combinatorial mutations (M12, M13, M23 and M123) also cause significant reduction in luciferase activities (*P<0.05). (H) Lentiviral shRNA-mediated knockdown of Klf2 results in decreased proliferation of PAC1 cells. Two different shRNA targeting distinct regions of Klf2 mRNA were used. Data are presented as mean±s.e.m. br, bronchus. Scale bars: 50 μm.
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