We find that during early lung development, around E13.5, Fgfr1 is moderately expressed in both the epithelium (Sox9, Sox2) and mesenchyme (Acta2, Adrp) ( Figures 1A, D and 2D, G ) and is upregulated in Sox9⁺ cartilage progenitors ( Figure 2A ). At E15.5, Fgfr1 is expressed at low levels in the epithelium and remains expressed in developing lipofibroblasts ( Figures 1B and 2H ), vascular smooth muscle cells, and cartilage progenitors ( Figures 2B, E, H ). By E18.5, Fgfr1 expression becomes relatively restricted to ADRP⁺ lipofibroblasts aka mesenchymal AT2 niche cells (MANC) ( Figure 2I ), vascular smooth muscle cells ( Figure 2F ) and tracheal cartilage ( Figure 2C ), with low expression levels in the conducting airway ( Figures 1C, F ).

Interestingly, we found Fgfr2 expression to be homogenously expressed in the lung epithelium at E13.5 ( Figures 1A, D ) as well as in airway smooth muscle cells ( Figure 2D ), and Sox9⁺ cartilage progenitors ( Figure 2A ). At E15.5, Fgfr2 is expressed in the epithelium with higher levels proximally vs distally ( Figures 1B, E, G ), airway smooth muscle cells ( Figure 2E ), and cartilage progenitors ( Figure 2B ). A downregulation of Fgfr2 signaling distally around E15.5 is required for the lung to transition from a branching program into an alveolar differentiation program; as we have previously demonstrated that overexpression of Fgf10 starting from E15.5 onwards prevents this transition by inducing distal epithelial Fgfr2 signaling (Volckaert et al., 2013b; Volckaert et al., 2019). Upregulation of Fgfr2 in the proximal epithelium at this stage also coincides with the differentiation of the basal cell lineage (Volckaert et al., 2013b; Balasooriya et al., 2017; Volckaert et al., 2017; Volckaert et al., 2019).

At E18.5 we find Fgfr2 to be highly expressed in the developing AT2 cells ( Figures 3B, G ), basal cells ( Figures 3C, G ), distal airway clubs at the BADJ ( Figures 1F and 3G ) and in cartilage cells ( Figure 2C ), with lower levels of expression in AT1 ( Figures 3A, G ) and proximal club cells ( Figures 3D, G ) and surrounding airway smooth muscle cells ( Figure 2F ). Furthermore, we find that at E18.5, there is no Fgfr2 expression in Foxj1⁺ ciliated cells or CGRP⁺ neuroendocrine cells ( Figures 3E, F ). These observations are consistent with our previous reports that overexpression of Fgf10 during late lung development blocks the differentiation of AT1 and ciliated cells in favor of AT2 and club or basal cells, respectively (Volckaert et al., 2013b; Volckaert et al., 2019).

During postnatal lung development, we found strong Fgfr2 expression in AT2 cells and strong Fgfr1 signaling in adjacent mesenchymal alveolar niche cells or lipofibroblasts ( Figures 4A–D, U–Y ). Interestingly, Fgfr2 expression was still present in AT1 cells but at lower level than in AT2 cells ( Figures 4E–H, Y ), which is consistent with an important role for Fgfr2 signaling in AT2 stem cell maintenance (Yuan et al., 2019). We further found Fgfr2 expression in club cells ( Figures 4M–P ), but not in ciliated cells ( Figures 4I–L ). Expression of Fgfr1 and 2 was low in airway and vascular smooth muscle cells ( Figures 4Q–T ). However, alveolar myofibroblasts exhibited modest Fgfr1 expression at P7 and P14 ( Figures 4Q, R, Y ), which is consistent with a lineage relationship with lipofibroblasts (Al Alam et al., 2015; El Agha et al., 2017; Li et al., 2018).

Next, to investigate the dynamic changes in both Fgfr1 and Fgfr2 expression after lung injury, we performed naphthalene and bleomycin injuries on adult mice at 8 weeks of age and traced the expression changes during lung regeneration. As expected, we found that Fgfr2 expression was pretty much gone in the airway at 3 and 7 days after naphthalene injury, consistent with a loss of club cells the main Fgfr2 expression cell type in the adult conducting airway ( Figures 5A–C, E–G and S1 ) (Volckaert et al., 2011). However, upon the return of club cells, Fgfr2 expression was gradually restored by 14 days after naphthalene injury ( Figures 5D, H ). We further found a modest increase in both Fgfr1 and Fgfr2 signaling in the airway smooth muscle cells upon naphthalene injury, consistent with an activation of this stem cell niche upon injury ( Figures 5I–L ) (Volckaert et al., 2011; Volckaert et al., 2013a; Lee et al., 2017). Remarkably, we found Fgfr2 upregulated in neuroendocrine bodies upon naphthalene injury, consistent with a role for Fgf10 signaling in regeneration of the airway epithelium by this distinct stem cell population ( Figures 5M–Q ) (Volckaert et al., 2011).

Lastly, we monitored the expression of Fgfr1 and Fgfr2 upon bleomycin injury. We found Fgfr2 to be higher in the distal airway club cells compared to proximal airway club cells at 3 and 6 weeks after bleomycin injury ( Figures 6A–C, P ). These findings are consistent with our previous report on the role of Fgf10-Fgfr2 signaling in alveolar epithelial regeneration by bronchial epithelial stem cells (Yuan et al., 2019). We found similarly high expression of Fgfr2 in regeneration AT2 cells upon bleomycin injury ( Figures 6D–F, P and S2 ) and in proximal neo-basal cells but significantly lower expression of Fgfr2 in more distal neo-basal cells ( Figures 6G–I, P ), consistent with our previous finding that Fgfr2 is required for the development of neo-basal cells upon bleomycin injury and that increased Fgf10 signaling can drive these cells along the AT2 cell lineage whereas reduced Fgfr2 signaling leads to their differentiation into AT1 cells (Yuan et al., 2019). Interestingly, we found Fgfr1 expression in myofibroblasts upon bleomycin injury ( Figures 6J–O ) consistent with them being derived from lipofibroblasts (El Agha et al., 2017; Yuan et al., 2019).

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead contact, SL (sdelanghe@uabmc.edu).

All mice were bred and maintained in a pathogen-free environment with free access to food and water. Both male and female mice were used for all experiments. Fgfr1^Cerulean (JAX 030708), Fgfr2^mCherry (JAX 030710) (Molotkov et al., 2017) mice were obtained from Jackson laboratories and crossbred to homozygosity. For bleomycin injury, adult 8 week old mice were intratracheally instilled with 50 µl bleomycin (1U/kg body weight for females, and 0.8U/kg body weight for males) as previously described (Yuan et al., 2019). For naphthalene injury, adult 8 week old mice were intraperitoneally injected with naphthalene dissolved in corn oil (325 mg/kg body weight for males and 300 mg/kg body weight for females) as previously described (Volckaert et al., 2011). All experiments were approved by the University of Alabama at Birmingham Institutional animal care and use committee.

All staining was done on paraffin sections of formalin-fixed lungs or tracheas. Immunofluorescent staining was performed with the following primary antibodies: goat anti-Scgb1a1 (1:200; clone T-18; sc-9772; Santa Cruz Biotechnology Inc.), rabbit anti-Scgb1a1 (1:500; WRAB-CCSP; Seven Hills Bioreagents), mouse anti-α-Actin (1:500; clone 1A4; sc-32251; Santa Cruz Biotechnology Inc.), chicken anti-GFP (1:250; GFP-1020; Aves Labs Inc.), rabbit anti-Keratin 5 (1:200; clone EP1601Y; MA5-14473; Thermo Fisher Scientific), chicken anti-Keratin 5 (1:500; clone Poly9059; 905904; BioLegend); rabbit anti-p63 (ΔN) (1:500; clone poly6190, 619002; BioLegend), 3862S; mouse anti-p63 (1:50; clone 4A4; CM163B; Biocare Medical), rabbit anti-RFP (1:200; 600-401-379; Rockland Immunochemicals Inc), mouse anti-RFP (1:200; sc-390909; Santa Cruz Biotechnology Inc.), rabbit anti-Sftpc (1:200; WRAB-9337; Seven hills bioreagents), mouse anti-Hop (1:100; Clone E-1; sc-398703; Santa Cruz Biotechnology Inc.), guinea pig anti-ADRP (Adipophilin) (1:200; 20R-AP002; Fitzgerald Industries), rabbit anti-Sox2 (1:1,000; WRAB-1236; Seven hills bioreagents), goat anti-Sox9 (1:500; AF3075; R&D systems), rabbit anti-CGRP (1:5,000; C8198; Sigma) and Mouse anti-Foxj1 (1:500, 14-9965-82, Invitrogen).

After deparaffinization, slides were rehydrated through a series of decreasing ethanol concentrations and antigens unmasked by either microwaving in citrate-based antigen unmasking solution (Vector Labs, H-3000) or by incubating sections with proteinase K (7.5 μg/ml) (Invitrogen, 25530-049) for 7 min at 37°C. Tissue sections were then washed in TBS with 0.1% Tween-20 and blocked with 3% bovine serum albumin (BSA), 0.4% Triton X-100 in Tris buffered saline (TBS) for 30 min at room temperature followed by overnight incubation of primary antibodies diluted in 3% BSA, 0.1% Triton X-100 in TBS. The next day, slides were washed in TBS with 0.1% Tween-20 and incubated with secondary antibodies diluted in 3% BSA, 0.1% Triton X-100 in TBS for 3h at room temperature. All fluorescent staining was performed with appropriate secondary antibodies from Jackson Immunoresearch, except for mouse anti-Hop (1:500; A-21125; Thermo Fisher Scientific). Slides were mounted using Vectashield with (Vector Labs, H-1200) or without DAPI (Vector Labs, H-1000) depending on immunostaining.

Tissue was imaged using a micrometer slide calibrated Zeiss LSM800 Laser scanning confocal microscope using ZEN imaging software. Images were processed and analyzed using Zen blue and Adobe Photoshop software. Average immunostaining intensity was quantified after segmentation and thresholding for pixel intensity.