Expression analysis of platelet-derived growth factor receptor alpha and its ligands in the developing mouse lung

Abstract

Activation of the platelet-derived growth factor receptor-α (PDGFRα) signaling pathway is critically important during lung alveogenesis, the process in lung development during which alveoli are formed from the terminal alveolar sacs. Several studies have aimed to characterize the expression patterns of PDGFRα and its two ligands (PDGF-A and -C) in the lung, but published analyses have been limited to embryonic and/or perinatal time points, and no attempts have been made to characterize both receptor and ligand expression simultaneously. In this study, we present a detailed map of the expression patterns of PDGFRα, PDGF-A and PDGF-C during the entire period of lung development, that is, from early embryogenesis until adulthood. Three different reporter mice were analyzed (Pdgfa^{ex4-COIN-INV-lacZ} , Pdgfc^tm1Nagy , and Pdgfra^{tm11(EGFP)Sor} ), in which either lacZ or H2B-GFP were expressed under the respective promoter in gene-targeted alleles. A spatiotemporal dynamic expression was identified for both ligands and receptor. PDGF-A and PDGF-C were located to distinct populations of epithelial and smooth muscle cells, whereas PDGFRα expression was located to different mesenchymal cell populations. The detailed characterization of gene expression provides a comprehensive map of PDGFRα signaling in lung cells, opening up for a better understanding of the role of PDGF signaling during lung development.

Keywords: Lung; platelet‐derived growth factor; platelet‐derived growth factor receptors; signal transduction.

Figure 1

Stages of lung development in mice. Time line indicating the different developmental stages: E‐embryonic stage (E9.5‐E12.5); P‐pseudoglandular stage (E12.5‐E15.5); C‐canalicular stage (E15.5‐E17.5); S‐saccular stage (E17.5‐P5); A‐alveolar stage (P5‐P30). Time points analyzed in this study are shown below in gray.

Figure 2

Expression patterns of Pdgfa, Pdgfc and Pdgfra in embryonic lung tissue. (A–J) X‐gal staining of Pdgfa ^lacZ (A–E) and Pdgfc ^lacZ lungs (F–J) counterstained with nuclear fast red. (B) Arrowheads point at weak X‐gal staining in the epithelium; (C) arrowheads point at X‐gal staining in airway smooth muscle cells; (E) arrowhead points at X‐gal staining in cell with AEC1 morphology and arrows point at cells with AEC2 morphology. (K–T) Pdgfra ^GFP lungs stained with antibodies against α‐sma (red) (K–T), E‐cadherin (yellow) and DAPI (blue) (P–T). (L, M) Arrowheads marks Pdgfra ^{GFP low} expression in α‐sma‐positive vascular smooth muscle cells and arrows mark Pdgfra ^{GFP high} cells coexpressing α‐sma. Time points: E10.5 (A, F, K,P); E12.5 (B, G, L, Q); E14.5 (C, H, M, R); E16.5 (D, I, N, S) and E18.5 (E, J, O, T). B – bronchus; Br – bronchiolus; Bv – blood vessel. Scale bars: 50 μm.

Figure 3

Expression patterns of Pdgfa, Pdgfc, and Pdgfra in postnatal lung tissue. (A–J) X‐gal staining of Pdgfa ^lacZ (A–E) and Pdgfc ^lacZ lungs (F–J) counterstained with nuclear fast red. (A–D) Arrows mark Pdgfa ^lacZ‐positive cells and arrowheads indicate extracellular X‐gal staining; (E) arrows mark X‐gal staining in bronchial epithelium and AEC2s, and arrowheads mark expression in SMCs; (G, H) arrowheads mark Pdgfc ^lacZ‐positive epithelial cells; (I, J) arrows mark Pdgfc ^lacZ positive SMCs. (K–T) Pdgfra ^GFP lungs stained with antibodies against α‐sma (red) (K–T), E‐cadherin (yellow) and DAPI (blue) (P–T). Time points: P1 (A, F, K, P); P5 (B, G, L, Q); P7 (C, H, M, R); P15 (D, I, N, S) and P60 (E, J, O, T). B – bronchus; Br – bronchiolus; Bv – blood vessel; Sc – saccule; A – alveolus Scale bars: 50 μm.

Figure 4

Quantification and immunostaining of Pdgfra ^GFP‐positive cells. (A) Amount of Pdgfra ^{GFP ‐}positive cells, as percentage of total number of cells in the alveolar region at different postnatal timepoints. Black bars indicate the total number of Pdgfra ^GFP ‐positive cells, and the gray bars present the Pdgfra ^{GFP high} (mean + SEM). (B, C) Cryo section of Pdgfra ^GFP P5 lung stained with antibodies against α‐sma (red). Arrowheads point at myofibroblasts in the secondary septae, expressing both α‐sma and Pdgfra ^GFP (green). (D, E) Cryo section of Pdgfra ^GFP P15 lung stained with antibodies against α‐sma. (F,G) Cryo section of Pdgfra ^GFP adult lung (P60) stained with Sftpc (red) showing the proximity of Sftpc⁺ and Pdgfra ^GFP‐positive cells (green). Scale bars: 20 μm.

Figure 5

Gene expression of Pdgfa, Pdgfc and Pdgfra. Quantitative PCR results (mean + SEM) of Pdgfa (A), Pdgfc (B), Pdgfra (C) at different developmental time points of wild‐type lungs. E12.5 was selected as a reference sample and values set at 1. Fold change was calculated through the Livak method, using 18s as an endogenous control and is represented on the y‐axis (2^−ΔΔCt). *P < 0.05, two‐way ANOVA, Tukey's multiple comparisons test.