REGULATION OF LUNG MESENCHYMAL CELLS BY EPITHELIAL WNT LIGANDS

The mesenchyme comprises of heterogeneous cell populations that support neighboring structures and are integral to intercellular signaling. Despite such importance, mesenchymal cells are poorly defined morphologically and molecularly. Their signaling regulation is not well understood, lagging their counterparts in the epithelial, endothelial, and immune lineages. Leveraging single-cell RNA-seq, three-dimensional imaging, and lineage tracing, first, we delineated different lung mesenchymal cell types and classified them into three-proximal-distal axes in the neonatal and adult murine lung. Each axis is associated to the structure they support – the endothelium, epithelium and interstitium. We also identified two subpopulations of neonatal lung myofibroblasts – ductal myofibroblasts that envelop alveolar ducts that persist into adulthood and alveolar myofibroblasts that undergo developmental apoptosis. Among lung mesenchymal subpopulations, we also found that myofibroblasts and pericytes experience canonical Wnt signaling. We know that the alveolar type 1 (AT1) epithelial cell signals via Vascular Endothelial Growth Factor A towards the lung vasculature. Given the large surface area of the AT1 cell, we hypothesized they had additional signaling roles. AT1 cells express canonical Wnt ligands, Wnt3a/7a. Epithelial double conditional knockout of Wnt3a/7a led in the neonatal lung to a drastic reduction in the expression of Platelet derived growth factor receptor alpha (PDGFRA) – a marker of myofibroblasts that is required for alveolus formation and is reduced in bronchopulmonary dysplasia (BPD), a chronic lung disease often associated with premature birth. Mutants also showed increased smooth muscle actin (ACTA2) expression in pericytes in the adult murine lung, a phenotype reminiscent to pulmonary hypertension. This phenotype was also confirmed by single cell RNA-seq, which additionally showed reduction in Axin2, a canonical Wnt target gene, specifically in the neonatal alveolar myofibroblasts and adult pericytes. Indeed, separate and independent conditional deletion of Ctnnb1 in alveolar myofibroblasts and pericytes recapitulated the reduction in PDGFRA expression and increased ACTA2 in pericytes, supporting that both myofibroblasts and pericytes directly respond to epithelial WNT3A and WNT7A. Establishing a novel link between the alveolar epithelium and mesenchymal cells sheds light on diverse functional roles of Wnt signaling in BPD and pulmonary hypertension therapies