Hedgehog and Platelet-derived Growth Factor Signaling Intersect during Postnatal Lung Development

Abstract

Normal lung development critically depends on HH (Hedgehog) and PDGF (platelet-derived growth factor) signaling, which coordinate mesenchymal differentiation and proliferation. PDGF signaling is required for postnatal alveolar septum formation by myofibroblasts. Recently, we demonstrated a requirement for HH in postnatal lung development involving alveolar myofibroblast differentiation. Given shared features of HH signaling and PDGF signaling and their impact on this key cell type, we sought to clarify their relationship during murine postnatal lung development. Timed experiments revealed that HH inhibition phenocopies the key lung myofibroblast phenotypes of <i>Pdgfa</i> (platelet-derived growth factor subunit A) and <i>Pdgfra</i> (platelet-derived growth factor receptor alpha) knockouts during secondary alveolar septation. Using a dual signaling reporter, <i>Gli1^lZ;Pdgfra^EGFP</i>, we show that HH and PDGF pathway intermediates are concurrently expressed during alveolar septal myofibroblast accumulation, suggesting pathway convergence in the generation of lung myofibroblasts. Consistent with this hypothesis, HH inhibition reduces <i>Pdgfra</i> expression and diminishes the number of Pdgfra-positive and <i>Pdgfra</i>-lineage cells in postnatal lungs. Bulk RNA sequencing data of Pdgfra-expressing cells from Postnatal Day 8 (P8) lungs show that HH inhibition alters the expression not only of well-established HH targets but also of several putative PDGF target genes. This, together with the presence of Gli-binding sites in PDGF target genes, suggests HH input into PDGF signaling. We identified these HH/PDGF targets in several postnatal lung mesenchymal cell populations, including myofibroblasts, using single-cell transcriptomic analysis. Collectively, our data indicate that HH signaling and PDGF signaling intersect to support myofibroblast/fibroblast function during secondary alveolar septum formation. Moreover, they provide a molecular foundation relevant to perinatal lung diseases associated with impaired alveolarization.