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

Lungs exhibit strikingly diverse epithelial architectures - from the branched airways of mammals to the sac-like lungs of lizards and the looped airways of birds. Across lineages, the pulmonary mesenchyme gives rise to smooth muscle that interacts with and shapes the underlying pulmonary epithelium. In mammals and lizards, pulmonary smooth muscle forms early and drives epithelial branching, whereas in birds it appears only after morphogenesis is largely complete. The developmental basis for this delay has remained unclear. Using comparative single-cell RNA sequencing, ATAC-sequencing, and imaging of mouse, anole, and chicken embryos, we found that smooth muscle in the chicken lung is transcriptionally similar to vascular, rather than visceral, smooth muscle. Strikingly, imaging revealed smooth muscle cells extending between the pulmonary vasculature and the epithelium, and surgical removal of these vessels prevented the formation of smooth muscle around the airways. The vascular transcription factor PITX2 was highly expressed in these cells and its knockdown markedly reduced smooth muscle differentiation. Taken together, these findings identify vascular smooth muscle as the developmental source of pulmonary smooth muscle in birds and establish PITX2 as a key regulator of this lineage transition, revealing an unexpected developmental and evolutionary link between the circulatory and respiratory systems.