Extended Data Fig. 1: Molecular characterization and mapping of the two alveolar capillary cell types.
a, Feature plots showing log-transformed expression of marker genes used to annotate five molecularly distinct clusters (see t-SNE plot in Fig. 1b) derived by unsupervised clustering of lung endothelial cells in the Tabula Muris scRNA-seq Smart-seq2 data13 for adult mouse lung. We identified two endothelial cell clusters as capillaries based on expression of carbonic anhydrases (Car4, Car14) that catalyse the conversion of bicarbonate to carbon dioxide41, Gpihbp1, a lipoprotein-binding protein that localizes to the luminal membrane of alveolar capillaries42, and lipoprotein lipase (Lpl), which is transported to the capillary lumen by Gpihbp143. b, Heat map showing log-transformed expression of selected cluster markers in individual pulmonary artery, vein, lymphatics and alveolar capillary (aCap, gCap) endothelial cells identified in the Tabula Muris Smart-Seq2 data13. c, Co-expression of alveolar capillary cell type markers Apln (aCap) and Aplnr (gCap) in a single alveolar capillary intermediate (IM) endothelial cell (arrow; marked by Cldn5) in adult mouse lung, as detected by smFISH. d, Co-expression of aCap markers (Apln and Ednrb) in a subset (aCap cells) of alveolar endothelial cells (marked by Cldn5) in adult mouse lung. e, Co-expression of aCap markers Ednrb and Car4 but not Aplnr, a gCap marker, in a subset (aCap cells) of adult mouse lung alveolar endothelial cells. e’, Boxed region from e at higher magnification. Filled arrowhead indicates aCap cell expressing high levels of Car4. Open arrowhead points to gCap cell expressing low levels of Car4. f, g, Co-expression of gCap markers Gpihbp1 (f) or H2-Ab1 (g), a major histocompatibility complex (MHC) class II gene, and Aplnr in a subset (gCap cells) of adult mouse lung alveolar endothelial cells. h, i, Quantification of the relative abundance (in % of capillary endothelial cells) of the two alveolar capillary populations (aCap, gCap) and rare cells (IM) that co-express aCap and gCap markers at the pleura and in intra-acinar regions (h), or in different lobes (i, left versus right cranial) in lungs from 3-month old mice. Data shown as mean ± s.d.; n = 500 cells scored per mouse; n = 3 mice; P values comparing cell type abundance by two-sided Wilcoxon rank sum test: aCap (h, 0.2; i, 0.2), gCap (h, 0.4; i, 0.2), IM (h, 0.7; i, 0.8). n.s., not significant. j, aCap (marked by Ednrb) and gCap (marked by Ptprb) cells in lungs from 3- and 24-month old mice. Endothelial cells are marked by Cldn5. k, l, Co-expression of tdTomato lineage label (asterisks) and either aCap marker Ednrb but not gCap marker Aplnr in an Apln-creER; Rosa26-tdTomato lung (k) or gCap marker Aplnr but not aCap marker Apln in an Aplnr-creER; Rosa26-tdTomato lung (l) harvested one month after mature aCap (k) or gCap (l) cells were lineage-labelled. Lineage-labelled cells (asterisks) continue to express the marker (Ednrb, aCap; Aplnr, gCap) of the labelled population. m, n, Quantification of percent of lineage-labelled cells in Apln-creER; Rosa26-tdTomato (m) or Aplnr-creER; Rosa26-tdTomato (n) lungs that continue to express the aCap marker, gCap marker, or markers of both cell types (IM) after 48 h, 1, 6, or 14 months (500–2,000 cells scored at each time point from multiple regions in each of two separate lobes from one lung). Blue (c–g, j–l), DAPI. Scale bars, 10 μm.
