Fig 2: Cell autonomous deletion of Fgfr2b in Sftpc-positive progenitors results in an increased proportion of RFP-labeled AT1 cells and mixed AT1 and AT2 cells. A Experimental design. Pregnant females carrying either control or experimental embryos were injected with TAM-IP at E14.5 and E15.5, and embryonic lungs were harvested at E18.5. B Control (n = 7) and experimental (n = 7) lungs were processed for FACS analysis and isolation of cells. EpCAMpos CD49fpos alveolar epithelial cells (AEC) were sorted, then the tomato RFPpos population was isolated. From these, AT1 and AT2 cells were isolated based on the expression of the AT1 marker T1α (Podoplanin). A decrease in the proportion of RFP-labeled alveolar epithelial cells (AECposRFPpos/AECpos) is observed in experimental (40.6% ± 2.57%) versus control lungs (48.64% ± 1.21%). A significant increase is seen in the proportion of RFP-labeled AT1 cells (AECpos RFPpos T1αpos/AECpos RFPpos) in experimental versus control lungs (85.5% ± 2.44% vs. 20.93% ± 0.64%); concomitantly, a significant decrease in labelled AT2 cells (AECpos RFPpos T1αneg/AECpos RFPpos) is observed in experimental versus control lungs (14.5% ± 2.44% vs. 79.07% ± 0.64%). (n = 7; ***p value < 0.001). C Immunofluorescent staining for PDPN (AT1 marker) and SFTPB (AT2 marker) in control Fgfr2b+/+ (a and b) and experimental Fgfr2bflox/flox (c and d) lungs. Single- and double-labeled cells are shown in ‘b’ (* = PDPNpos, # = RFPpos, + = SFTPBpos/RFPpos). Triple-labeled cells are shown in ‘d’ (arrowheads = PDPNpos/SFTPBpos/RFPpos). Scale bar: (a, c) 30 µm, (b, d) 7.5 µm. D Quantification of ‘C’ shows (a) decreased proportion of RFP-labeled AT2 (SFTPB-positive) cells in experimental versus control lungs (32.23% ± 2.94% vs. 54.34% ± 3.33%); (b) an increased proportion of RFP-labeled AT1 (PDPNpos) cells in experimental versus control samples (19.81% ± 2.01% vs. 4.78% ± 0.17%); (c) as well as an increased proportion of RFP-labeled cells expressing both AT1 and AT2 markers in experimental versus control lungs (24.89% ± 2.88% vs. 15.59% ± 0.13%). (n = 3; *p value < 0.05, **p value < 0.01)

Fig 3: Postnatal inactivation of Igf1r from lung secondary crest myofibroblast cells.(A) Schematic of the experimental protocol. (B) Hematoxylin and eosin (H&E) staining of lung sections from control (a–c) and Gli1CreERT2 mutant (d-f) mice and their morphometric measurements by mean linear intercept (MLI) (g–i) and peri/area ratio (j–l) at postnatal day 7 (PN7), PN14, and PN30. See Figure 2—figure supplement 1 for the definition and calculation of these indices. (C) Immunostaining of lung sections from control (a–d) and mutant (e–h) mice for elastin (a,e), ACTA2 (b,f), AQP5/HOPX (c,g), and SFTPC/PDPN (d,h), and the comparison of the number of AT1 (i) and AT2 (j) cells between the control and mutant. Quantitative data was presented as mean values +/-SD with data for each experiemntal group collected from eight lobes from four different lungs. p-Value: * stands for 0.05–0.01, ** for 0.01–0.001, *** for <0.001, n.s. for not significant. The same designation is used throughout the paper. Scale bar: 100 um in B and 25 um in C.

Fig 4: Characterization of FRC phenotypes in lymph nodes of pdgfrb-cre::Lama5fl/fl and WT (Lama5fl/fl) mice immunized with OVA and analyzed at 1 week after immunization(A) FRCs were sorted from lymph nodes of WT mice and CD45+CD31negPDGFrecβ+gp38 high and low populations were analyzed by qPCR for markers of follicle border specific FRCs;(B) the same analyses were performed on sorted CD45+CD31neg PDGFrecβ+gp38low FRCs from OVA immunized pdgfrb-cre::Lama5fl/fl mice and WT controls. Data in A and B are expressed as relative fold difference and are means ± SD of 2 experiments performed with 8 pdgfrb-cre::Lama5fl/fl and 6 WT mice. Data were analyzed using a Mann Whitney U test, ∗∗p <0.01; ∗∗∗p <0.001.(C–E) (C) Sorted CD4+PD-1+CXCR5+ Tfh-cells from pdgfrb-cre::Lama5fl/fl and WT lymph nodes were analyzed by qPCR and (D) results were verified by flow cytometry of CD4+T-cells and quantified in (E). Mean fluorescence intensity (MFI) in (E) are means ± SD of 4 experiments performed with 4 pdgfrb-cre::Lama5fl/fl and 4 WT mice. Data were analyzed by Mann Whitney U tests; ∗∗∗p <0.001.See also Figure S6.