Single cell RNA analysis uncovers the cell differentiation and functionalization for air breathing of frog lung.

The evolution and development of vertebrate lungs have been widely studied due to their significance in terrestrial adaptation. Amphibians possess the most primitive lungs among tetrapods, underscoring their evolutionary importance in bridging the transition from aquatic to terrestrial life. However, the intricate process of cell differentiation during amphibian lung development remains poorly understood. Using single-cell RNA sequencing, we identify 13 cell types in the developing lungs of a land-dwelling frog (Microhyla fissipes). We elucidate the differentiation trajectories and mechanisms of mesenchymal cells, identifying five cell fates and their respective driver genes. Using temporal dynamics analyses, we reveal the gene expression switches of epithelial cells, which facilitate air breathing during metamorphosis. Furthermore, by integrating the published data from another amphibian and two terrestrial mammals, we illuminate both conserved and divergent cellular repertoires during the evolution of tetrapod lungs. These findings uncover the frog lung cell differentiation trajectories and functionalization for breathing in air and provide valuable insights into the cell-type evolution of vertebrate lungs.