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. 2023 Sep 13;14(1):5630.
doi: 10.1038/s41467-023-41309-3.

A single-cell atlas of West African lungfish respiratory system reveals evolutionary adaptations to terrestrialization

Ruihua Zhang #  1   2   3 , Qun Liu #  2   3   4 , Shanshan Pan #  2   3 , Yingying Zhang  2   3 , Yating Qin  2   3 , Xiao Du  2   3   5 , Zengbao Yuan  1   2   3 , Yongrui Lu  6 , Yue Song  2   3 , Mengqi Zhang  2 , Nannan Zhang  2   3 , Jie Ma  2   3 , Zhe Zhang  7 , Xiaodong Jia  8 , Kun Wang  9 , Shunping He  6 , Shanshan Liu  5   7 , Ming Ni  5   7 , Xin Liu  5 , Xun Xu  5   10 , Huanming Yang  5 , Jian Wang  5 , Inge Seim  11   12 , Guangyi Fan  13   14   15
Affiliations

A single-cell atlas of West African lungfish respiratory system reveals evolutionary adaptations to terrestrialization

Ruihua Zhang et al. Nat Commun. .

Abstract

The six species of lungfish possess both lungs and gills and are the closest extant relatives of tetrapods. Here, we report a single-cell transcriptome atlas of the West African lungfish (Protopterus annectens). This species manifests the most extreme form of terrestrialization, a life history strategy to survive dry periods that can last for years, characterized by dormancy and reversible adaptive changes of the gills and lungs. Our atlas highlights the cell type diversity of the West African lungfish, including gene expression consistent with phenotype changes of terrestrialization. Comparison with terrestrial tetrapods and ray-finned fishes reveals broad homology between the swim bladder and lung cell types as well as shared and idiosyncratic changes of the external gills of the West African lungfish and the internal gills of Atlantic salmon. The single-cell atlas presented here provides a valuable resource for further exploration of the respiratory system evolution in vertebrates and the diversity of lungfish terrestrialization.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Single-cell transcriptional profiles of West African lungfish lung and gill under freshwater and terrestrialized conditions.
a Experimental workflow in this study. AE denotes terrestrialization; FW, freshwater. L, lung; G, gill. b Lung cell landscape of 53,605 cells from terrestrialized and freshwater lungfish. Cells are shown by Uniform Manifold Approximation and Projection and color-coded by cluster cell type. Each dot represents a cell, and different colors are associated with specific cell types. Vasc_Endo, vascular endothelial cell; Vasc_SMC, vascular smooth muscle cell; SMC, smooth muscle cell; Un_epithelial cell, unclassified epithelial cell; Un_lymphoid cell, unclassified lymphoid cell. c Gill cell landscape of 87,347 cells from terrestrialized and freshwater lungfish. Cells are shown by UMAP and color-coded by cluster cell type. Each dot represents a cell, and different colors are associated with specific cell types. NK cell_like, Natural killer cell_like; Un_epithelial cell, unclassified epithelial cell; Un_immune cell, unclassified immune cell. d Cell type identification of six classical lung cell type markers (see Supplementary Fig. 1c for overall markers). Markers are shown by density plots. Values indicate the max density. The color scale is relative, ranging from 0 to highest density. e Cell type identification of six classical gill cell type markers (see Supplementary Fig. 1d for overall markers). Annotated as in (d). f Confirmation of scRNA-Seq cell type annotation results by H&E stanning and fluorescence microscopy image of African lungfish lung. Green, digoxigenin-labeled marker genes probes amplified using FITC-TSA; blue, DAPI (scale bar =  500 μm). (1) fibromuscular wall; (2), air sacs. On the H&E image, the red solid circle represents alveolar epithelial cells; red rhombus represents macrophage cells; red circle represents the lymphoid nodes. Each slide was repeated independently 3–5 times. Scalebar, 500μm. g Confirmation of scRNA-Seq cell type annotation results by H&E stanning and fluorescence microscopy image of African lungfish gill. Annotated as in (f). Fl, filament; Cr, cartilage; Wight arrows, lamella. Each slide was repeated independently 3–5 times.
Fig. 2
Fig. 2. Cellular and molecular features of lungfish lungs during terrestrialization.
a Schematics of the electron transport chain with differentially expressed genes (DEGs) in lung alveolar epithelial cells indicated. b The average expression levels of DEGs (see a) in alveolar cells during terrestrialization (AE; in maroon) and in freshwater (FW; in salmon) by bar plot. c Comparisons of selected DEGs between freshwater and terrestrialized lung cell types by bubble plot. AE denotes terrestrialization (red bubbles); FW, freshwater (blue bubbles). Circle size reflects the percentage of cells within a cell type which express the specific genes. d Enriched KEGG pathway of AE macrophage up regulated DEGs. The enrichment analysis was generated via the Enrichr web server using Fisher exact test, and the Benjamini-Hochberg (BH) was used for correction for multiple hypotheses testing. P values are indicated on the x-axis. e Comparison of cellular interactions among all cell types in terrestrialized lungfish lung (AE_lung, left) and freshwater lungfish lung (FW_lung, right) by net plots. Each node represents a kind of cell type, links mean the legend-receptor interactions and the width of link is in direct proportion to the LR pair numbers. f Enriched biological processes of LR pairs in the lungfish lung. Generated using the Enrichr web server. Annotated as in Fig. 2d. g Schematics of the transmembrane coreceptor NRP1 and its ligands VEGF and SEMA3A. h The expression of selected LR pairs in lung cell types from freshwater (FW) and terrestrialized (AE) lungfish. Circle (AE) or triangle (FW) size reflects the percentage of cells within a cell type which express the specific genes. The color of circles/triangles indicates the average expression of the LR pair. P values were calculated by CellphoneDB, which means the likelihood of cell-type specificity of a given receptor–ligand pairs. Source data are provided as a Source Data file.
Fig. 3
Fig. 3. Difference in gills between freshwater and terrestrialized condition.
a Comparison of selected DEGs between freshwater and terrestrialized gill cell types by bubble plot. Annotated as in Fig.2c. b Schematics of the reactive oxygen species pathway differentially expressed genes (DEGs) in gill proliferative cells indicated. c The average expression levels of DEGs (see Fig.2b) in gill proliferative cells during terrestrialization (AE; in dark blue) and in freshwater (FW; in light blue).
Fig. 4
Fig. 4. Cell-cell communication of gills between freshwater and terrestrialized condition.
a Comparison of cell-cell communications between cell types in terrestrialized lungfish gill (left) and freshwater lungfish gill (right) by heatmap. Color means the log-normalized numbers of CellPhoneDB ligand-receptor pairs (LR pairs) b Enriched biological processes of LR pairs in the lungfish gill. The enrichment analysis was generated via the Enrichr web server using Fisher exact test, and the Benjamini-Hochberg (BH) was used for correction for multiple hypotheses testing. P values are indicated on the x-axis. c The expression of selected LR pairs in gill cell types of freshwater (FW) and terrestrialized (AE) lungfish. Annotated as in Fig. 2h. Source data are provided as a Source Data file.
Fig. 5
Fig. 5. Cell type evolution of the lung and gill.
a Pairwise cell type similarities of the zebrafish swim bladder, lungfish lung, mouse lung, and human lung by sankey plot. The top 5% highest Kullback–Leibler divergence (KDL) values are indicated as arches connecting cell types for each pair. b Conserved expression in alveolar cells of the human lung, lungfish lung, and mouse lung. Violin plots are including 57 orthologous genes (>1.3 log2 fold-change). Each dot represents a gene, and normalized average expression values are shown. c Enriched biological processes of 57 orthologous genes in b. The enrichment analysis was generated via the Enrichr web server using Fisher exact test, and the Benjamini-Hochberg (BH) was used for correction for multiple hypotheses testing. P values are indicated on the x-axis. d The expression of selected DEGs in lungfish-zebrafish-human-mouse comparisons in the lungfish lung. Circle size reflects the percentage of cells within a cell type that expresses the specific genes. The color of circles indicates the average expression of a DEG. e The expression of lung cell type DEGs in lungfish compared to human. Mean values are shown. DEGs with a q-value ≤ 0.05 and fold change ≥3 are shown. f Cell–cell similarities between the gills of the lungfish and Atlantic salmon. The top 5% highest Kullback–Leibler divergence (KDL) values are indicated as arches connecting cell types. g Violin plots of 40 orthologous genes (>1.3 log2 fold-change) with conserved expression in MRCs of the salmon gills, lungfish gills.Annotated as in (b). h The expression of DEGs of lungfish-Atlantic salmon gill comparisons in the lungfish gill. The x-axis of volcano plot shows the log2 fold-change, the y-axis represents the percent of lungfish and salmon gill cells that express each gene (calculating the Δ percentage difference). Source data are provided as a Source Data file.
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