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

Abstract

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.

Fig. 1. Cellular diversity of lungs in 4 developmental stages.

a A schematic of the basic workflow for the pulmonary cell landscape in M. fissipes using the 10× Genomics platform. b Ultrastructural dynamics of alveoli showing the proportional variations in the major cell types, AEC alveolar epithelial cell, EC endothelial cell, MSC mesenchymal cell, RBC red blood cell; scale bar, 10 μm. c Dot plot showing the expression levels of marker genes for pulmonary cells. d UMAP showing unbiased clustering results of all of the filtered cells in lung. e UMAP visualizing the predicted pulmonary cell trajectory.

Fig. 2. Construction of differentiation trajectories of MSCs.

a UMAP visualization of MSCs. b UMAP visualizing the predicted differentiation trajectory of MSC lineages. c Circular plot visualizing the predicted cell fates of MSC lineages. d UMAP showing the cell fate probabilities of MSC lineages. e–g Dot plots showing the putative driver genes of different MSC fates, each dot represents a gene, colored by mean expression across all cells.

Fig. 3. Heterogeneity and temporal molecular dynamics of pulmonary epithelial cells in 4 developmental stages.

a UMAP visualization of pulmonary epithelial cells. b GO enrichment of feature genes in pulmonary epithelial cells (adjusted p < 0.001). c Ultrastructural characters of AECs and ciliated cells. Scale bar, 5 μm. Violin plots showing the temporal dynamic expression of feature genes regulating cell growth and proliferation (d) and genes involving in cell function (e) in pulmonary epithelial cells with development. (***) indicates a significant difference among the expression level in different stages (p < 0.001).

Fig. 4. The peculiarity of AECs in adult M. fissipes.

Ultrastructural characters of alveoli in adult mouse (a) and M. fissipes (b). BV blood vessel, LB lamellar body, MV microvillus, N nucleus, scale bar, 10 μm. c FISH presenting the expression of AHNAK (green) and SFTPB (red) in AECs of M. fissipes, co-expression showed yellow fluorescence. Scale bar, 50 μm. d The UMAP plots presenting co-expression of AHNAK and LGALS3 (green) with SFTPB (red) in AECs of M. fissipes based on scRNA-seq data.

Fig. 5. Cell heterogeneity of pulmonary ECs.

a UMAP visualizing the feature genes of pulmonary ECs. b Venn diagram showing the gene number uniquely expressed and shared by two types of ECs. The blue, orange, and brown area represent the gene number uniquely expressed in EC_Ls, EC_Vs, and shared by two types of ECs, respectively. Bar plots showing the GO enrichment of feature genes uniquely expressed in EC_Ls (c) and EC_Vs (d) and shared by two types of ECs (e).

Fig. 6. Cell type evolution of the lung.

Circle plot showing the similarity of cell lineages between Microhyla fissipes and Xenopus laevis (a), mouse (b), and human lung (c), respectively. d Sankey plot showing the pairwise cell type similarities of the X. laevis, M. fissipes, mouse, and human lung. The different colors of dots represent various cell type. The illustrations of X. laevis, mouse, and human are created with MedPeer (medpeer.cn).