Tissue-specific responses to transpulmonary pressure promote branching morphogenesis of the mouse lung

Abstract

During embryogenesis, the mammalian lung undergoes successive rounds of epithelial tube branching to form a complex tree-like architecture. Transmural pressure regulates the rate of airway branching morphogenesis, with higher pressures increasing the rate of branching events. Because many genes with retinoic acid (RA) response elements (RAREs) in their proximal enhancer region are differentially expressed at high transmural pressure, we hypothesized that RA signaling may be activated by transmural pressure during development. Here, we used mutant mice expressing a RARE reporter to investigate whether the mechanosensitive transcriptional regulator Yap, which regulates differentiation in many tissues throughout development, plays a role in activating RA signaling in response to pressure. Combining bulk and single-cell transcriptomic datasets, we examined tissue specific responses to pressure and Yap deletion. These analyses revealed that epithelial markers are upregulated at higher pressures, while markers for both the sub-epithelial and sub-mesothelial mesenchyme are downregulated. Next, we modulated luminal pressure in ex vivo lung culture and used immunostaining approaches to identify tissue-specific responses to pressure. We found that proliferation at the branch tips of the airway epithelium is elevated at high pressure, in agreement with our bioinformatic findings. In the pulmonary mesenchyme, patterning of distinct cell populations is altered by changes in transmural pressure. Furthermore, we found that RA signaling activity is increased in the epithelium of lungs cultured at high pressure, and this pressure-dependent change may be modulated by deletion of Yap from the pulmonary mesenchyme and/or epithelium. Together, these results provide insight into how the mechanical microenvironment of the developing lung impacts the process of branching morphogenesis.