Fig. 5: Fgfr2 pathway prevents AT2 reprogramming to AT1 after birth.

a scRNAseq of adult AT2 cells. Expression of Fgfr2, downstream transcription factor Etv5, and target genes indicates pathway remains on after birth. AT2, AT2 markers; Ubiq, ubiquitously-expressed genes. b, c AT2 in adult (PN60) Sftpc-CreER;Rosa26-mTmG mouse tamoxifen-induced to label AT2 (GFP) and co-stained for Fgfr2 (b) or phosphorylated MAP kinase (pMAPK) to show pathway activity (c). Blue, DAPI counterstain. Bars, 10 µm. d Quantification of b, c. Nearly all labeled cells (GFP⁺) express AT2 marker (Muc1) and Fgfr2, and show Fgfr2 activity (pMAPK⁺). n = 300 GFP⁺ cells scored (three animals). e Fgfr2 ligand expression (scRNAseq) in lung stromal cells. f AT2 in adult Sftpc-CreER;Rosa26-mTmG mouse tamoxifen-induced to label AT2 (GFP⁺) and co-stained for Fgfr2 (isoform iiib) ligand-binding domain to show Fgfr2 ligands (ligands identified in Supplementary Fig. 2). Blue, DAPI. Bar, 10 µm. g smFISH (Fgf7, Fgf10, Sftpc) in adult (PN60) lung. Cell neighboring AT2 co-expresses Fgf7 (white dots) and Fgf10 (red dots) (enlarged in inset). Blue, DAPI; arrowhead, AT2. Bar, 5 µm. h, i Quantification of g showing the density of Fgf-expressing and AT2 cells (h, ***p = 4.8 × 10⁻⁵, Student’s two-sided t-test, data shown as mean ± SD) and distance between them (i, ***p = 2.9 × 10⁻⁵⁰, Mann Whitney, boxplot shows minimum, maximum, first quartile, second quartile (median), and third quartile). n = 182 AT2, 103 Fgf⁺ cells scored (four animals). (Values indicate Fgf availability and how many AT2 it supports, and how far each Fgf-expressing cell must extend (or ligand diffuse) for support). j–o Alveolar region of LyzM-Cre;Rosa26-mTmG;Fgfr2^fl/+ (j–l) or LyzM-Cre;Rosa26-mTmG;Fgfr2^fl/fl (m–o) mice (ages indicated) immunostained for Cre reporter (mTmG), AT2 (Muc1), and macrophage (Mac2, distinguishes GFP-labeled macrophages) markers. LyzM-Cre becomes active in AT2 in early postnatal life, activating the GFP reporter and deleting Fgfr2^fl. Control GFP^pos AT2 cells carry a wild-type Fgfr2 allele (Fgfr2^fl/+, j–l) and remain AT2 (inset in l), whereas those lacking Fgfr2 (Fgfr2^fl/fl, m–o) convert to AT1 (arrowheads, inset in o). Bar, 50 µm. p–r Quantification (mean ± SD) of j–o showing increasing Cre activity in AT2 in early postnatal life (p) and identities of GFP^pos cells in control (q) and following Fgfr2 loss (r). n = 300 GFP^pos cells scored (three animals) per timepoint/condition; *p = 0.01 for (p) and ***p = 0.0002 for (r) (Student’s two-sided t-test, time series end vs. start).