Canonical BMP-pathway is active in the developing vascular and sub-epithelial smooth muscle networks.
A) Confocal images of tissues from different developmental stages stained with antibodies to eGFP and CD31 (parenchyma and blood-vessels), and eGFP and αSMA (blood-vessels and large-airways). Nuclei were counterstained with DAPI. Representative eGFPpos cells in the different tissue compartments are shown with arrows. Green arrows depict eGFPpos cells, whereas yellow arrows depict eGFPpos-CD31pos or eGFPpos-αSMApos cells. A white arrow in the P1 vessel indicates a rare eGFPpos-αSMApos vascular smooth-muscle cells. “Aw” defines airways and “V” vessels. Scale bar: 20 µm. B) Whole E12 lung-explants, cultured on Nuclepore membranes, were treated with vehicle, SB431542, or LDN193189. Upper panel shows transmitted light images of representative explants. Lower panel shows eGFP expression in the same explants. C) Confocal images of tissue-sections from lung-explants treated with vehicle, SB431542 or LDN193189 for 8-hours and stained with antibodies to eGFP, CD31 and αSMA. Nuclei were counter-stained with DAPI. D) Quantitative-PCR analysis of eGFP, Id1, FGF10, BMP4 and VEGFα mRNA expression in vehicle or inhibitor-treated lung explants. Values represent mean ± SEM of six SB431542-treated, seven LDN193189-treated and thirteen vehicle treated, individually analyzed explants per group, compared using one-way analysis of variance (ANOVA) with Bonferoni's post-hoc analysis. *P<0.05, **P<0.01 (vehicle vs inhibitor treated groups) and #P<0.05, ##P<0.01, ###P<0.001 (SB vs LDN treated groups).