Expression of canonical WNT/β-CATENIN signaling components in the developing human lung

Abstract

Background: The WNT/β-CATENIN signaling cascade is crucial for the patterning of the early lung morphogenesis in mice, but its role in the developing human lung remains to be determined. In this study, expression patterns of canonical WNT/β-CATENIN signaling components, including WNT ligands (WNT2, WNT7B), receptors (FZD4, FZD7, LRP5, LRP6), transducers (DVL2, DVL3, GSK-3β, β-CATENIN, APC, AXIN2), transcription factors (TCF4, LEF1) and antagonists (SOSTDC1) were examined in human embryonic lung at 7, 12, 17 and 21 weeks of gestation (W) by real-time qRT-PCR and in situ hybridization.

Results: qRT-PCR analysis showed that some of these components were gradually upregulated, while some were significantly downregulated from the 7 W to the 12 W. However, most components reached a high level at 17 W, with a subsequent decrease at 21 W. In situ hybridization showed that the canonical WNT ligands and receptors were predominantly located in the peripheral epithelium, whereas the canonical WNT signal transducers and transcription factors were not only detected in the respiratory epithelium, but some were also scattered at low levels in the surrounding mesenchyme in the developing human lung. Furthermore, Western blot, qRT-PCR and histological analysis demonstrated that the β-CATENIN-dependent WNT signaling in embryonic human lung was activated in vitro by CHIR 99021 stimulation.

Conclusions: This study of the expression patterns and in vitro activity of the canonical WNT/β-CATENIN pathways suggests that these components play an essential role in regulation of human lung development.

Figure 1

Real-time qRT-PCR was performed to examine the mRNA expression levels (mean ± sem) of canonical WNT/β-CATENIN signaling components, including WNT ligands (WNT2 , WNT7B ), WNT receptors ( FZD4 , FZD7 , LRP5 , LRP6 ), WNT transducers ( DVL2 , DVL3 , GSK-3β , β-CATENIN , APC , AXIN2 ), WNT transcription factors ( TCF4 , LEF1 ) and WNT inhibitors or antagonists ( SOSTDC1 ) in the developing human lung at 7 W, 12 W, 17 W and 21 W (W: Weeks of gestation) (A-D). Data were normalized to the average mRNA level of β-actin at 7 W. Statistical difference is indicated by asterisks (* P < 0.05, ** P < 0.01).

Figure 2

Expression of canonical WNT/β-CATENIN signaling ligands in the developing human lung. ( A- D) Morphogenesis of the early human lung development at 7 W, 12 W, 17 W and 21 W (W: Weeks of gestation). WNT2 ( E- H) and WNT7B ( I- L) expression was detected in the epithelium of human lungs at 7 W, 12 W, 17 W and 21 W. Scale bars = 100 μm in A- L.

Figure 3

Expression of canonical WNT/β-CATENIN signaling receptors in the developing human lung. The expression of canonical WNT signaling receptors, including FZD4 ( A- D), FZD7 ( E- H), LRP5 ( I- L) and LRP6 ( M- P), was clearly detected in the distal part of human lung epithelium at 7 W, 12 W, 17 W and 21 W (W: Weeks of gestation). Scale bars = 100 μm in A- P.

Figure 4

Expression of canonical WNT/β-CATENIN transducers in the developing human lung. Sections of the human embryos at the gestational ages of 7 W, 12 W, 17 W and 21 W (W: Weeks of gestation) showed particular expression of canonical WNT signal transducers DVL2 ( A- D), GSK-3β ( I- L) and APC ( Q- T) in the distal epithelium. However, the expression of WNT signal transducers DVL3 ( E- H), CTNNB1 (also β-CATENIN, M- P) and AXIN2 ( U- X) was detected mainly in the distal epithelium and also weakly in the mesenchyme of human embryonic lungs at the gestational age of 7 W, 12 W, 17 and 21 W. Scale bars = 100 μm in A- X.

Figure 5

Expression of canonical WNT/β-CATENIN signaling transcription factors in the developing human lung. ( A- D) Expression of canonical WNT signaling transcription factor TCF4 was detected in both the distal epithelium and the lung mesenchyme of human embryos at 7 W, 12 W, 17 W and 21 W (W: week of gestation). ( E- H) LEF1 staining of in situ hybridization showed a strong reactivity in the distal epithelium of the lung at 7 W, 12 W, 17 W and 21 W in human embryos. ( I- L) Sections with SOSTDC1 staining showed particular expression patterns in the distal lung epithelium of the embryos at 7 W, 12 W, 17 W and 21 W (W: week of gestation). ( M- P) Negative control for in situ hybridization in the developing human lung. Scale bars = 100 μm in A- P.

Figure 6

Activity of canonical WNT/β-CATENIN signals induced by CHIR 99021 in the developing human lung. Human lung tissue explants obtained at 15 weeks of gestation were exposed to 0, 5 and 10 μM CHIR 99021 in vitro for 72 h. ( A- B) Western blot analysis showed expression of β-CATENIN in human lung tissues. Results are presented as the mean ± sem of three independent experiments. * P < 0.05, ** P < 0.01. ( C) qRT-PCR analysis detected a dose-dependent increase in WNT signaling transcription factors and target genes following exposure to CHIR 99021. Results are presented as the mean ± sem of three independent experiments. * P < 0.05, ** P < 0.01. ( D- K) Histology examination showed obvious morphogenetic changes of the airway epithelial cells associated with CHIR 99021 treated lung tubes. Scale bars: D- G =100 μm; H- K = 20 μm.