Collection of fetal lung tissues was carried out at the embryonic (E11 and E12) pseudoglandular (E13.5 and E14.5), canalicular (E17) and saccular (E19.5) stages. Noon of the day of the vaginal plug was considered embryonic day (E) 0.5. Additionally, tissues were collected on post-natal days 1 (P1), 15 (P15) and 2 months after birth (adult). All embryonic and P1 lungs were removed surgically, cleaned from the surrounding tissues and placed in 4% paraformaldehyde (Merck, 104004) in PBS for 24 hours at 4uC. For analysis of P15 and adult BRE mice the right lung-lobes were used for RNA/Protein isolation, whereas the left lungs were gently perfused with a mixture of 4% PFA:OCT (2:1) using a 20G catheter (Abbocath G717-A01, 4535-20), the bronchus was ligated and placed in 4% PFA for 24 hours at 4uC. Thereafter, the tissues were placed in PBS with 30% sucrose for 24 hours at 4uC, washed with PBS, embedded in OCT (Shandon Cryomatrix) and kept at 280uC until use. Cryostat sections 6-10 mm (Leica CM3050S) were placed on polylysine or SuperFrost Plus slides (Menzel Glaser), kept at room temperature for 3 hours with silica gel (Merck, 101969) and then stored at 280uC. The primary antibodies used for immunostaining were: goat anti-CC10 (Santa Cruz, SC9773), rat anti-PECAM1 (CD31) (BD-Biosciences, 550274), rat anti-VEGFR2 (eBiosiences 14-5821), rabbit anti-SpC (Chemicon, AB3786), rabbit anti-CGRP (Sigma-Aldrich, C8198), mouse anti-a smooth muscle actin-Cy3 (Sigma-Aldrich, C6198), Syrian hamster anti-T1a (DSHB 8.1.1), Sheep anti-BrdU (Fitzgerald, 20-BS17), mouse anti-Foxj1 (generous gift of Dr. S. Brody), mouse biotinylated anti-Muc5ac (Abcam, ab79082), anti-SM22alpha (Abcam, ab14106) and chicken anti-GFP (Abcam, ab13970). The secondary antibodies used were: donkey anti-goat Texas Red (Jackson Immuno-research, 705-076-147), donkey antichicken FITC (Jackson Immuno-research, 703-096-155), goat anti-rat AlexaFluor594 (Molecular Probes, A11007), goat anti-rat AlexaFluor647 (Molecular Probes, A21247), goat anti-Syrian hamster AlexaFluor568 (Molecular Probes, A21112), goat anti-Syrian hamster AlexaFluor647 (Molecular Probes, A21541), donkey anti-rabbit Texas-Red (Jackson Immuno-research, 711-076-152), donkey anti-sheep AlexaFluor568 (Molecular Probes, A21099) and donkey anti-rabbit Cy5 (Jackson Immuno-research, 711-176-152). The slides were mounted using the ProLong Gold Antifade Reagent with DAPI (Molecular Probes, P36931). Images were captured with a Leica DMRA2 fluorescent microscope equipped with a Leica DFC320 and DFC350 FX digital cameras and a Leica TCS-SP5 confocal microscope (Leica Microsystems, Wetzlar, Germany). Image analysis was performed using Adobe Photoshop CS3, ImageJ 1.41 and Volocity LE. pSmad1/5/8 immuno-staining Tissue sections prepared as described in the previous section were treated 1.5% H 2 O 2 in MetOH for 30 min at RT in the dark to inactivate endogenous peroxidise. After blocking with 2% normal donkey serum (Sigma D9663) in TBS-Tween 0.3% for 2 hours at room temperature, the sections were incubated with rabbit anti-pSmad1/5/8 (Chemicon, ab3848) antibody overnight at 4uC in TBS-Tween 0.3% and then incubated for 1 hour at room temperature with the secondary anti-Rabbit HRP (Santa Cruz, SC2301). The slides were developed using DAB chromogen (DAKO K3468) development according to the manufacturer's instructions. Normal rabbit IgG fraction was used as control primary antibody (isotype control). Nuclei were counterstained with Mayer's Hematoxylene.