Fig. 4

Longitudinal single-cell RNA-seq reveals the heterogeneity of epithelial subtypes and alveolar epithelial regeneration after long-term exposure to coal dust. A The tSNE plot shows the changes in the distribution of the subtypes of epithelial cells 9 months after coal dust-mediated lung injury. B Differentially expressed genes (DEGs) from scRNA-seq were identified, and the top 50 DEGs are shown. C Volcano plots highlight differentially expressed genes in the coal dust-exposed and control pulmonary cells. Red dots indicate gene expression upregulation in coal dust-exposed mice, while blue dots indicate downregulation after coal exposure. Labels were added to DWFb1, Krt5, and the top 20 most DEGs. D Gene ontology (GO) classification provides putative gene functions for pulmonary cells in coal-exposed mice. E Single-cell annotation and unsupervised migration clustering identified nine subtypes of epithelial cells. F Based on the lung epithelial cell analysis, the annular heat map shows the significant DEGs out of the top 50 DEGs. The single-cell sequencing data revealed that these top 50 genes were differentially expressed in alveolar dual potent progenitor cells and type 2 alveolar epithelial cells. G CHIL1 and SFTPC overexpression in-coal exposed lungs were identified by immunohistochemical staining that compared the coal-exposed lung tissues to the control lungs. H The proliferating epithelial subsets expressed APOC1, CXCL15, and IL33, covering the primary epithelial cells with CHILA1, LYZ, and HBEGF. Cell markers were used to identify clusters as represented in the tSNE plot. I Using pseudotime ordering analysis, we successfully constructed the pulmonary epithelial cell lineage as a differentiation trajectory. Each branch shows a single-cell state. The top left plot is marked with developmental time, and the right plot is marked with cell states