Poster Q11 - Uncovering the effects of transmural pressure on differentiation in the embryonic lung

Introduction: The airways of the mammalian lung are generated by branching of epithelial tubes into the surrounding mesenchyme, a process that is influenced by the transmural pressure across the epithelium. The airways are comprised of several distinct cell types including pulmonary neuroendocrine cells (PNECs), which are the first epithelial cell to differentiate in the embryonic lung. PNECs are initially specified in a salt-and-pepper pattern throughout the airway epithelium, and then undergo an epithelial-mesenchymal transition (EMT) that enables a unique form of cell migration, called slithering, which leads to the clustering of PNECs into neuroepithelial bodies (NEBs) at branch points in the tree. PNECs and NEBs are thought to serve as oxygen, chemical, and mechanical sensors in the lung, and the numbers of these cells are elevated in pathological conditions associated with altered transmural pressure, including those related to premature birth. Here, we combine transgenic mice and microfluidic systems to understand the role of transmural pressure in the regulation of PNEC differentiation and NEB clustering. Specifically, we combine timelapse imaging analysis and quantitative measurements to reveal whether transmural pressure affects differentiation, EMT, slithering, or all three events in PNEC fate.

Materials and

Methods: We isolated lungs at embryonic day (E)14 and either fixed them immediately or culture them for 24 or 48 hours. In one series of experiments, lungs were cultured in microfluidic chest cavities to directly vary the transmural pressure across the epithelium from 20 Pa to 400 Pa. In a complementary series of experiments, lungs were cultured on filters in the presence of forskolin, which promotes fluid secretion out of the lung and indirectly increases transmural pressure. Samples were then fixed and immunostained for Ascl1 and Prox1 to label PNECs and NEBs. In a separate series of experiments, explants isolated from Ascl1CreER/+;Rosa26mTmG embryos were used for timelapse imaging analysis to monitor the effects of pressure on slithering and the formation of NEBs. We also performed bulk RNA-sequencing and single cell RNA-sequencing analyses to uncover how the expression of PNEC markers changes in response to alterations in transmural pressure.

Results, Conclusions, and Discussions: RNA-sequencing analysis revealed that low-pressure conditions increase the expression of markers for PNECs, suggesting that differentiation of these cells is inversely correlated with transmural pressure. Consistently, immunofluorescence analysis for the PNEC markers Ascl1 and Prox1 revealed an inverse correlation between the number of PNECs and magnitude of transmural pressure. These data indicate that transmural pressure regulates the first step of PNEC specification in the developing epithelium. Future work will define how physical and molecular signals downstream of transmural pressure regulate the differentiation of PNECs.

Acknowledgements (Optional): This work was supported in part by grants from the National Institutes of Health (HL164861, HD099030, HD111539). SB was supported by a postdoctoral fellowship from the Momental Foundation and SB is The Shurl and Kay Curci Foundation Awardee of the Life Science Research Foundation.