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Mesodermal progenitor-specific deletion of Pdgfrα. A. Quantitative PCR (Q-PCR) showed PDGF signaling target genes are decreased, indicating an overall functional repression of PDGFA signaling pathway in E14.5 Pdgfra Dermo1 lungs. n = 2 pairs of separate lungs, repeated twice or thrice. B. Q-PCR showed repression of myofibroblast-related genes in Pdgfra Dermo1 lungs. n = 2 pairs of separate lungs, repeated thrice for each. Error bars show SEM. (PPTX 68 kb)

Mesodermal Alk5 deficiency increased cell apoptosis. The number of TUNELpos cells as determined by previously described methods [12]. Aâ D. TUNEL assay showed increased number of TUNELpos cells in the Alk5 Dermo1 lungs at E18.5. E. Quantification of the relative number of TUNELpos cells (green) per 4,000 total cells. Error bars show standard deviation. *P

Mesodermal-specific Alk5 inactivation causes pulmonary hypoplasia. A–D. Gross morphology of E16.5 control (A and B) and Alk5 Dermo1 (C and D) lungs. E–J. PAS (E, H, G and J) and H&E (F and I) staining of E18.5 control (E–G) and Alk5 Dermo1 (H–J) lungs. Arrows in I indicate a thicker alveolar wall in Alk5 Dermo1 lungs; Arrows in J indicate robust PAS staining in Alk5 Dermo1 lungs. Scale bars: H = 100 μm; I = 10 μm, J = 20 μm. (PPTX 1736 kb)

Quantification of smooth muscle (SM) thickness and areas covered by smooth muscle in control and Pdgfrα Dermo1 lungs at E18.5. αSMA immunohistochemical stained lung sections were collected by random sampling using the 40× objective from three pairs of control and Pdgfrα Dermo1 mutant lungs at E18.5. The thickness and coverage of SM were quantified. A. Quantification of SM thickness. The arithmetic mean thickness of the SM cell layer was determined by volume of αSMApos compartment, measured by counting all points intercepting the airway epithelium and αSMApos compartment. n = 3. B. Quantification of SM coverage. The percentage of SM airway coverage was determined by assessing the percentage of the airway epithelium circumference in contact with number of αSMApos cells. n = 3. Error bars show the standard deviation. *P

Mesodermal Alk5 deficiency inhibits epithelial cell differentiation and disrupts vasculogenesis. A–H. Immunohistochemistry (IHC) for Acetylated-tubulin (A and E), CC10 (B and F), pro-SPC (C and G) and T1a (D and H) in E18.5 control and Alk5 Dermo1 lungs showing decreased expression of the latter genes in Alk5 Dermo1 lungs. I. Q-PCR confirmed inhibition of epithelial cell markers in Alk5 Dermo1 lungs, n = 2–9 pairs of separate lungs. J and L: IHC for GFP and FLK1 showing decreased GFP+/FLK1+ cells in E14.5 Alk5 Dermo1 lungs. K and M: IHC showed decreased CD34+ cells in E14.5 Alk5 Dermo1 lungs. N–Q. IHC showing decreased of PECAM1+ cells in E18.5 Alk5 Dermo1 lungs. Arrows indicate large blood vessels appearing intact. R. Quantitative PCR confirmed decreased Pecam1, Flk1, and Flt1 mRNAs in E13.5 mutant lungs. n = 3 pairs of separate lungs. Error bars show standard deviation. *P

Quantification of sub-mesothelial mesenchyme (SMM) size. Hematoxylin and eosin-stained lung tissue sections were prepared and random images were collected under 40× objective from both control and Alk5 Dermo1 embryos at E12.5 and E13.5. The various dimensions that were measured included boundaries of the SMM compartments, as delineated on each photomicrograph, from mesothelium to the mesenchyme wrapped around the epithelium (SEM). The regions encompassing the SMM were measured in Photoshop by dividing each region at random of six points to manually calculate the length from mesothelium to SEM (μm). Sample size, n = 3 separate lung tissue sections. Error bars show standard deviation. *P

Primary antibodies used in western blots and immunohistochemistry. (DOC 37 kb)

Dermo1-cre-mediated mesodermal progenitor-specific deletion of Alk5. A–C. Dermo1-cre expression pattern in E14.5 Dermo1-cre;mTmG lungs. Dermo1-cre-mediated recombination (green fluorescence) was located in the mesenchyme surrounding the trachea (A), bronchi (B), bronchioles and blood vessels (C), not in the epithelial cells. D. Deletion of Alk5 exon 3 by crossing Alk5 flox/flox to Dermo1-cre mice. E. Deletion of Alk5 was validated by PCR with genomic DNA. F. Western blot analysis using total protein from E16.5 control and Alk5 Dermo1 lungs. n = 3. β-ACTIN was used as a control. G–J. Immunohistochemistry for ALK5 (G and I) and PAI-1 (H and J) showed lost or decreased ALK5 and PAI-1 (Arrows in I and J) in Alk5 Dermo1 lungs. Scale bars: C, J = 20 μm. (PPTX 925 kb)

Quantification of Oil Red O storage. Lung tissue sections from E18.5 control and Alk5 Dermo1 lungs were analyzed by staining with Oil Red O for assessment of LIF differentiation. Oil Red O stained sections were viewed and random images were collected under 40× objective, and were analyzed by imaging using a previously described protocol [49], n = 3. The density of Oil Red O stained cells was then calculated and plotted as shown. Error bars show the standard deviation. *P