Rac1 modulates mammalian lung branching morphogenesis in part through canonical Wnt signaling

Abstract

Lung branching morphogenesis relies on a number of factors, including proper epithelial cell proliferation and differentiation, cell polarity, and migration. Rac1, a small Rho GTPase, orchestrates a number of these cellular processes, including cell proliferation and differentiation, cellular alignment, and polarization. Furthermore, Rac1 modulates both noncanonical and canonical Wnt signaling, important pathways in lung branching morphogenesis. Culture of embryonic mouse lung explants in the presence of the Rac1 inhibitor (NSC23766) resulted in a dose-dependent decrease in branching. Increased cell death and BrdU uptake were notably seen in the mesenchyme, while no direct effect on the epithelium was observed. Moreover, vasculogenesis was impaired following Rac1 inhibition as shown by decreased Vegfa expression and impaired LacZ staining in Flk1-Lacz reporter mice. Rac1 inhibition decreased Fgf10 expression in conjunction with many of its associated factors. Moreover, using the reporter lines TOPGAL and Axin2-LacZ, there was an evident decrease in canonical Wnt signaling in the explants treated with the Rac1 inhibitor. Activation of canonical Wnt pathway using WNT3a or WNT7b only partially rescued the branching inhibition. Moreover, these results were validated on human explants, where Rac1 inhibition resulted in impaired branching and decreased AXIN2 and FGFR2b expression. We therefore conclude that Rac1 regulates lung branching morphogenesis, in part through canonical Wnt signaling. However, the exact mechanisms by which Rac1 interacts with canonical Wnt in human and mouse lung requires further investigation.

Keywords: Rac1; branching morphogenesis; canonical WNT; lung.

Fig. 1.

Rac1 is expressed in mouse and human developing lungs. A and B: qRT-PCR showing the expression of RAC1 in human fetal lungs between gestational weeks 10 and 21 (A) n = 3 at least, and in the developing mouse lung between embryonic day E11.5 and E18.5 (B), n = 3 for each time point. C–E: immunofluorescent staining showing active Rac1 (green) and DAPI (blue) in 12.5 (C), 15.2 (D), and 18 wk (E) gestational age human fetal lung. F–H: fluorescent staining using the fusion protein GST-PAK-PBD (red) and DAPI (blue) on 12.5 (F), 15.2 (G), and 18 wk (H) human fetal lung. I–J: immunofluorescent staining showing active Rac1 (green) and DAPI (blue) in E13 (I) and E14 mouse lung (J). K: negative control in absence of active-Rac1 antibody and only secondary antibody on E13 mouse lung. L and M: staining using the fusion protein GST-PAK-PBD (red) and DAPI (blue) on E13 (L) and E14 (M) mouse lung. N: negative control in presence of anti-GST and secondary antibody only on 12.5 wk human fetal lung. Staining was performed on slides from at least 3 independent samples. Scale bar is 50 μm.

Fig. 2.

Rac1 inhibition decreased embryonic mouse lung branching in a dose-dependent manner. Whole-mount view of E12.5 mouse lung explants at 0 h (A, C, E, and G) and following 48 h in culture (B, D, F, and H) in the absence (A and B) or presence of 6.25 (C and D), 12.5 (E and F), and 25 μM (G and H) of Rac1 inhibitor NSC23766. I: quantification of fold change in number of buds (no. of buds at t = 48 h/no. of buds at t = 0 h). Data are represented as means ± SE of at samples from at least 4 independent litters; n = 5 at least; *P = 0.0281; ***P = 0.0005.

Fig. 3.

Rac1 inhibitor EHop-016 impairs embryonic mouse lung branching. Whole-mount view of E12.5 mouse lung explants at 0 h (A, C, E) and following 48 h in culture (B, D, F) in the absence (A and B) or presence of 8 μM (C and D) and 10 μM (E and F) of Rac1 inhibitor EHop-016. G: quantification of fold change in number of buds in explants treated with 8 μM EHop-016 vs. control DMSO (no. of buds at t = 48 h/no. of buds at t = 0 h). H: fold change in Axin2 expression in explants treated with 8 μM EHop-016 compared with controls. Data are represented as means ± SE of at samples from at least 4 independent litters. *P ≤ 0.01.

Fig. 4.

Rac1 inhibition leads to increased cell proliferation and cell death in the lung mesenchyme. A and B: GST-PAK-PBD staining on sections treated with 25 μM NSC23766 (B) vs. controls (A). C and D: TUNEL staining on E12.5 mouse lung sections cultured for 48 h in absence (C) or presence of 25 μM Rac1 inhibitor (D). E and F: BrdU staining on E12.5 mouse lung sections cultured for 48 h in absence (E) or presence of 25 μM Rac1 inhibitor (F). White lines delineate epithelial structures. G: quantification of BrdU-positive cells in either the epithelium or mesenchyme as percentage to the total number of cells within the appropriate each compartment in controls (white bars) and treated explants (gray bars). Data are represented as means ± SE of at least 6 independent samples, P = 0.002. H–K: cultured isolated epithelial buds from E11.5 mouse lungs at t = 0 (H and J) and t = 48 h (I and K) in presence of FGF10 alone (H and I) or FGF10 + NSC23766 (J and K). L–O: cultured isolated mesenchymal explants from E11.5 mouse lungs at t = 0 (L and N) and t = 48 h (M and O) in presence of FGF9 alone (L and M) or FGF9 + NSC23766 (N and O); n = 4 independent samples. P and Q: TUNEL staining on cultured isolated mesenchymal explants from E11.5 mouse lungs cultured for 48 h treated with FGF9 (P) or FGF9 + NSC23766 (Q). R: Ecad staining on cultured isolated mesenchymal explant from E11.5 mouse lung cultured for 48 h. S: Ecad and Acta2 staining on cultured isolated epithelial bud from E11.5 mouse lung at t = 0. Scale bars are 100 μm in A, C, P, and S and 50 μm in E.

Fig. 5.

Rac1 regulates smooth muscle cells and Fgf10 expression. A and B: Acta2 staining in control (A) and NSC23766-treated (B) explants following 48 h in culture. C: quantification by qRT-PCR of Acta2, Fgf10, Spry2, Spry4, Etv4, Etv5, Fgfr2b, and Fgfr2c of control and treated explants following 48 h in culture. Data are represented as means ± SE of at least 7 independent samples; *P ≤ 0.05. Scale bar is 50 μm.

Fig. 6.

Rac1 inhibition alters lung vasculogenesis ex vivo. A and B: LacZ staining on Flk1-LacZ reporter explants cultured in absence (A) or presence (B) of Rac1 inhibitor. C and D: immunofluorescence for CD31 (red) of untreated (C) and Rac1 inhibitor treated (D) lung explants; blue is DAPI. E: quantification of Vegfa by qRT-PCR in control and treated lung explants. Data are represented as means ± SE of at least 7 independent experiments; *P = 0.0007. Scale bar is 50 μm.

Fig. 7.

Rac1 inhibition decreases canonical Wnt signaling. A and B: lung explants from TOPGAL reporter mice cultured in absence (A) or presence (B) of Rac1 inhibitor. Lung explants from Axin2-LacZ reporter mice cultured in absence (C) or presence (D) of Rac1 inhibitor. E: quantification of Wnt inhibitors Dkk1 and Wif1 by qRT-PCR in control and treated lungs. Data are represented as means ± SE of 7 independent experiments; *P = 0.0018. F: LacZ staining of E13.5 Axin2-LacZ lung and its WT littermate.

Fig. 8.

Wif1 inactivation is not sufficient for rescuing lung branching due to Rac1 inhibition. A–D: E12.5 lung explants from Wif1^−/− mice cultured in absence (A and B) or presence (C and D) of NSC23766 at t = 0 (A and C) and t = 48 h (B and D). E: quantification of the fold change in number of buds between 48 h and 0 h of control and NSC23766-treated lungs. Data are represented as means ± SE of 8 independent experiments. *P ≤ 0.05.

Fig. 9.

Wnt activation partially rescues lung branching defects caused by Rac1 inhibition. A–L: E12.5 WT lung explants at t = 0 (A, C, E, G, I, and K) and t = 48 h (B, D, F, H, J, and L) cultured in absence of NSC23766 (A and B), in presence of NSC23766 (C and D), in presence of NSC23766 and 200 ng WNT3a (E and F), in presence of 200 ng of WNT3a alone (G and H), in presence of NSC23766 and 140 ng WNT7b (I and J), and in presence of 140 ng of WNT7b alone (K and L). M: quantification of the fold change in number of buds in the explants after 48 h in culture in presence or absence of NSC23766 with or without WNT3a. Data are represented as means ± SE of 6 independent experiments; *P ≤ 0.05. N: quantification of the fold change in number of buds in the explants after 48 h in culture in presence or absence of NSC23766 with or without WNT7b. Data are represented as means ± SE of 6 independent experiments; *P ≤ 0.05. O–R: LacZ stain of Axin2-LacZ lung explants cultured for 48 h in presence of NSC23766 (O), NSC23766 and WNT3a (P), NSC23766 and WNT7b (Q), or WNT3a alone (R). S and T: immunofluorescence staining for β-catenin (red) on control (S) and NSC23766-treated explants (T). U: relative expression of β-catenin in control and treated explants as assessed by qRT-PCR. Results are expressed as means ± SE of 6 independent experiments normalized to control values. Scale bar in R is 50 μm.

Fig. 10.

Rac1 inhibition impairs human lung branching in vitro. A–D: Dissected 11.1-wk gestational age distal edges of 11 wk gestational age human lung explants at t = 0 (A, C) cultured on air liquid interface in absence (A, B) or presence of NSC23766 (C, D) for 48 h (B, D). E and F: TUNEL staining on sections from paraffin-embedded control explants (E) and NSC23766-treated explants (F) after 48 h in culture. G and H: immunofluorescence for CD31 on control (G) and treated sections (H). I: graph showing fold change in number of branches at 48 h compared with t = 0 h. J: graph illustrating the fold change in AXIN2, FGF10, WIF1, VEGFA, and FGFR2b expression in treated explants normalized to controls. Results in I and J are expressed as means ± SE of explants from 4 independent samples. Scale bars are 50 μm. *P ≤ 0.05.