Mechanical forces and smooth muscle differentiation in the developing lung

Introduction: The airway epithelial tree is constructed in the embryo through a stereotyped and recursive process of branching morphogenesis, which is guided by both biochemical as well as mechanical signaling between the lung epithelium and its surrounding mesenchyme. Here, we study the formation of terminal bifurcations, which occur when the tip of an elongating epithelial tube bifurcates into two daughter branches. The bifurcation process is initiated and sculpted by cells in the adjacent mesenchyme, which differentiate into smooth muscle cells at the future cleft site and then wrap around the tip of the epithelium and squeeze it into two daughter branches. This positioning of smooth muscle differentiation is critical for the final pattern of the epithelial tree, but it is unclear how the site of smooth muscle differentiation is specified. Here, we test the hypothesis that mechanical signals from the growing epithelium promote smooth muscle differentiation.

Materials and

Methods: We combine fluorescent oil droplets with transgenic reporter animals to quantitively analyze patterns of endogenous stresses and to monitor the tissue flows in the mesenchyme that lead to smooth muscle differentiation and subsequent bifurcation of the epithelium. We use immunofluorescence analysis of the basement membrane and interstitial matrix and EdU incorporation assays to infer spatial patterns of material properties and cell proliferation, respectively. We also create a theoretical framework to describe how the process of branch elongation couples with mechanical forces and changes in material properties to pattern mechanical signals that might promote smooth muscle differentiation.

Results, Conclusions, and Discussions: Surprisingly, EdU analysis reveals that there are no obvious spatial patterns in cell proliferation that correlate with smooth muscle differentiation or epithelial branching. In contrast, immunofluorescence analysis for components of the basement membrane and interstitial matrix are consistent with spatial gradients in the material properties of the mesenchyme in regions where smooth muscle will differentiate. We combine these experimental results with theory to understand the physical mechanisms that promote branch elongation and specify the site of terminal bifurcation. Our data suggest that these patterns create a mechanical microenvironment that facilitates the differentiation of mesenchymal cells into smooth muscle cells. Further work will define the biochemical signaling coordinating branch elongation and smooth muscle differentiation at the bifurcation site.

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.