Origins of smooth muscle and evolutionary specializations of the pulmonary mesenchyme in the vertebrate lung

Evolution has generated a beautiful diversity of epithelial architectures in the lungs of terrestrial vertebrates, from the tree-like lungs of mammals, to the sac-like lungs of reptiles, to the looped airways of birds. The physical mechanisms that generate pulmonary epithelia of such different shapes are just beginning to be uncovered. Pulmonary smooth muscle plays stage-dependent roles in shaping the epithelium during lung development in the mouse, anole, and chicken. We hypothesized that differences in the timing and spatial patterns of smooth muscle differentiation correspond to distinct patterns of gene expression in the pulmonary mesenchyme. To test this hypothesis and investigate differences in pulmonary smooth muscle patterning across terrestrial vertebrates, we generated scRNA-seq datasets from lungs of mouse, anole, and chicken embryos. These data revealed that smooth muscle regulators are expressed in similar patterns in mouse and anole lungs, in which epithelial branches are sculpted by smooth muscle, but diverge in chicken lungs, in which the epithelium branches in the absence of smooth muscle. Pulmonary smooth muscle differentiates from the lung mesenchyme in mouse and anole. Surprisingly, however, bioinformatic and experimental evidence suggest that pulmonary smooth muscle is derived from vascular smooth muscle in chicken. Furthermore, our data revealed differences in the timing and extent of smooth muscle innervation across species. These findings highlight how terrestrial vertebrates have evolutionarily repurposed similar morphogenetic motifs to build lungs that suit their energetic needs. ### Competing Interest Statement The authors have declared no competing interest. * αSMA : α-smooth muscle actin; CK : cytokeratins; E : embryonic day; scRNA-seq : single cell RNA sequencing; UMAP : uniform manifold approximation and projection;