Fig. 1: Expression of Fgfr2 and its ligands during alveolar differentiation.

a Expression of Fgfr2 and the four other receptor genes most selectively expressed in alveolar type 2 (AT2) cell lineage (“Receptors”) from single-cell RNA sequencing (scRNAseq) analysis of distal (alveolar) epithelial cells from embryonic day 18.5 (e18.5) mouse lung. Cells (columns) are arranged in developmental pseudotime (BP, bipotent progenitors, center; AT1 lineage to the left; AT2 lineage to the right) determined as described in ref. 11 by expression of alveolar lineage markers like the four shown (“Lineage”) including two that restrict from BP to AT2 lineage (BP/AT2, Sftpc) or to AT1 lineage (BP/AT1, Rage). (Note due to the very high expression of Sftpc in bipotent progenitors, there is a temporal lag in the extinction of the transcripts in newly-differentiating AT1 cells and thus some nascent AT1 cells co-express Sftpc and Rage.) Ubiq, ubiquitously-expressed control genes. Heat map, mRNA expression level. b Expression of Fgfr2b ligand genes in distal (alveolar) endothelium and mesenchyme from scRNAseq of e18.5 lung. c e17.5 lung immunostained for Fgfr2 (red), bipotent progenitor (BP) and AT1 marker podoplanin (Pdpn, green), and epithelial marker E-cadherin (E-cad, white). Boxed regions, close-ups, and split channels at the right of the bipotent progenitor (BP) and developing AT2 lineage cell (AT2 lin). Note in BP close-up BPs are cuboidal, Pdpn-expressing cells. In AT2 lin closeup, Fgfr2 (red) remains on in developing AT2 cells (arrowhead) but downregulated in neighboring developing AT1 cells, and although BP/AT1 marker (Pdpn) is still detected it too will soon be downregulated as developing AT2 cell matures (see Supplementary Fig. 1a). Bars, 50 µm (left panel), 10 µm (close-ups). Biological triplicates were analyzed. d e17.5 lung stained with the Fgfr2 (isoform iiib) ligand-binding domain fused to human IgG₁ domain to show Fgfr2b ligands (red), and co-stained for Pdpn (green), and AT2 marker mucin1 (Muc1, white). Fgfr2b ligands are detected in nearby mesenchymal cells (asterisk) and diffusely around developing alveolar epithelial cells. Bar, 10 µm. Repeated in biological triplicate. e Schematic showing inferred signaling from Fgf ligand-expressing mesenchymal cell (fibroblast) to nearby Fgfr2-expressing nascent AT2 cell during alveologenesis. pre-AT1, nascent AT1 cells that downregulated Fgfr2. All experiments were repeated at least three times.