Fig. 6: Fgfr2 signaling continuously prevents adult AT2 apoptosis.

a (Left) Time course showing the proportion of AT2 (Muc1⁺) cells labeled with GFP in control (LyzM-Cre; Rosa26-mTmG Fgfr2^fl/+) or Fgfr2 deleted (LyzM-Cre; Rosa26-mTmG; Fgfr2^fl/fl) lungs in postnatal life (control, extension of Fig. 5p, mean values ± SD). No further accumulation of GFP^pos AT2 occurs in Fgfr2 deleted lungs (dashed line) after PN20, despite continued LyzM-Cre activity (AT2 labeling) apparent in control (solid line). n = 3955 AT2 (3 animals/genotype). (Right) Time course showing identities of GFP^pos cells following Fgfr2 loss (extension of Fig. 5r). After a rapid increase in AT1 due to AT2 reprogramming in the juvenile period (through PN20), no further accumulation of GFP^pos AT1 or AT2 occurs. n = 300 GFP^pos cells scored (3 animals/condition and timepoint, mean ± SD). b Close-up of the alveolar region in adult (PN60) LyzM-Cre;Rosa26-mTmG;Fgfr2^fl/fl mouse stained for apoptosis marker cleaved Caspase-3 (cCaspase-3). Note GFP^pos AT2 deleted for Fgfr2 undergoing apoptosis (asterisk) but not unlabeled control AT2 nearby (arrowhead). Bar, 10 µm. c–f MASTR system for the conditional, complete deletion of Fgfr2 in AT2. c AAV-Sftpc-Flp virus (top) expresses Flp from Sftpc promoter. MASTR allele (bottom), following Flp-mediated deletion (at frt, triangles) of Pgk-neo-pA cassette, constitutively expresses GFP-Cre fusion. d Scheme. Two weeks after AAV-Sftpc-Flp instillation into lungs of adult Fgfr2^fl/fl;Rosa26^mTmG/MASTR mice to induce constitutive GFP-Cre and complete deletion of Fgfr2^fl/fl in AT2, lungs were immunostained (GFP, Muc1, Fgfr2, cCaspase-3). e Alveolar regions from Fgfr2^fl/+ control (top) or Fgfr2^fl/fl (bottom) mice treated as in (d). Note the reduction in GFP^pos AT2 (Muc1^pos) (bottom). Bar, 25 µm. f Quantification of AT2 loss in (e). n = 890 Muc1^pos cells scored/condition (three experiments, mean ± SD). g, h Quantification (h) of Fgfr2 immunostaining (n = 100 GFP^pos cells scored/condition, three experiments, mean ± SD). Nearly all (96%) GFP^pos AT2 remaining after MASTR-mediated Fgfr2^fl/fl deletion (e, f) have not fully lost Fgfr2. Micrograph (g) shows an example of rare GFP^posFgfr2^neg AT2, which were all rounded and extruded into the lumen. Bar, 10 µm. i, j Quantification (j) of cCaspase-3 immunostaining (n = 470 GFP^pos cells scored/condition, mean ± SD). Note that 36% of GFP^pos AT2 cells remaining after MASTR-mediated Fgfr2^fl/fl deletion were cCaspase-3^pos, indicating apoptosis initiated (micrograph, i). Bar, 10 µm. k–n Acute inhibition of Fgfr2 signaling by FIIN-1. k Scheme for co-instillation of FIIN-1 and fluorescent marker WGA-405 into lungs of adult Sftpc^CreER/+;Rosa26^mTmG/+ mice tamoxifen(Tam)-treated to label AT2 then analyzed over 2 weeks. Bar, 200 µm. l Lung regions exposed to FIIN-1 (WGA-labeled, left) and shown merged (right) with mTmG fluorescence (AT2 lineage GFP-labeled, other cells tdTomato (mT)-labeled). Bar, 250 µm. m WGA-labeled alveolar regions exposed 2 h to the vehicle (top) or FIIN-1 (bottom). FIIN-1-exposed AT2 cells (GFP^pos) undergo apoptosis (cCaspase-3^pos, arrowheads). Bar, 20 µm. n Kinetics of AT2 apoptosis in (m). In vehicle control, <2.5% of AT2 were cCaspase-3^pos (n = 650 cells scored/condition, three experiments, mean ± SD). No conversion of labeled AT2 to AT1 was detected during this period (n = 390 cells scored/condition, three experiments) (see also Suppl. Fig. 7). o–q Effect of acute Fgfr2 inhibition in AT2 cells by Fgfr2iiib-Fc protein. o Scheme. After Tam-induction of adult Sftpc^CreER/+;Rosa26^mTmG/+ mice to GFP-label AT2, Fgfr2iiib-Fc (or vehicle) and WGA-405 were co-instilled and lungs subsequently stained for cCaspase-3. p WGA-labeled alveolar regions from vehicle control (top) and Fgfr2iiib-Fc (bottom) instilled lungs. Bar, 25 µm. q Quantification of p. n, cells scored, six experiments (mean ± SD). ***p = 4.3 × 10⁻⁴ for (f), **p = 3.4 × 10⁻³ for (j), and ***p = 2.5 × 10⁻⁴ for (q) (Student’s two-sided t-test) (panels f, j, q).