FGFR2b signalling restricts lineage-flexible alveolar progenitors during mouse lung development and converges in mature alveolar type 2 cells

Ethical statement and husbandry

Animal experiments and harvesting organs and tissues from wildtype and mutant mice following euthanasia using pentobarbital were approved at Justus Liebig University Giessen by the federal authorities for animal research of the Regierungspraesidium Giessen, Hessen, Germany (Approved Protocol GI 20/10 Nr. G 84/2016).

All mice used to generate experimental and control embryos were housed in a specific pathogen-free (SPF) environment with free access to food and water. Up to five females were housed together, while males were housed singly. Females between 9 and 12 weeks of age were used to generate embryos.

In vivo mouse models

Experiments to inhibit FGFR2b ligands were conducted using a previously described and validated inducible dominant negative mouse model: ROSA26rtTA/rtTA; Tg(tet(o)sFgfr2b)/+ (B6-Cg-Gt(ROSA)26Sortm1.1(rtTA,EGFP)Nagy Tg(tetO-Fgfr2b/lgh)1.3Jaw/sbel) [13, 15]. These mice were generated by crossing Rosa26rtTA/rtTA mice with Rosa26rtTA/rtTA; Tg(tet(o)sFgfr2b)/+ mice to obtain experimental (Rosa26rtTA/rtTA; Tg(tet(o)sFgfr2b)/+) and littermate control (Rosa26rtTA/rtTA; +/+) embryos. This model employs a reverse tetracycline transactivator (rtTA) under the transcriptional control of the ubiquitous Rosa26 locus. Upon administration of doxycycline, the rtTA binds to the tetracycline operator (tetO), inducing the transcription of a soluble dominant negative form of Fgfr2b (sFgfr2b), which is secreted from cells and acts to sequester all FGFR2b ligands in the extracellular matrix.

Cell-autonomous Fgfr2b deletion and lineage labelling in alveolar progenitors was achieved by using a cell-type specific CreERT2 recombinase combined with a tandem dimer (td)Tomatoflox reporter and an Fgfr2bflox knock-in line. Upon administration of tamoxifen, CreERT2 translocates to the nucleus, recombining the loxP-flanked stop cassette upstream of the tdTomato construct, as well as excising the IIIb exon in the Fgfr2 gene. Targeting of AT1 progenitors was achieved by creating HopxCreERT2/+; Fgfr2bflox/flox; tdTomatoflox/flox embryos (Stock Hopxtm2.1(cre/ERT2)JoeFgfr2tm1DsnGt(ROSA)26Sortm9(CAG-tdTomato)Hze/sbel). Experimental litters were obtained by crossing HopxCreERT2/+; Fgfr2bflox/flox; tdTomatoflox/flox mice with Hopx+/+; Fgfr2bflox/flox; tdTomatoflox/flox mice, whereas control litters were obtained by crossing HopxCreERT2/+; Tomatoflox/flox mice with tdTomatoflox/flox mice. Targeting of AT2 progenitors was achieved by creating SftpcCreERT2/+; Fgfr2bflox/flox; tdTomatoflox/flox embryos (STOCK Sftpctm1(cre/ERT2,rtTA)HapFgfr2tm1Dsn Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/sbel) [24]. Experimental litters were obtained by crossing SftpcCreERT2/+; Fgfr2bflox/flox; tdTomatoflox/flox mice with Sftpc+/+; Fgfr2bflox/flox; tdTomatoflox/flox mice, whereas control litters were obtained by crossing SftpcCreERT2/+; tdTomatoflox/flox mice with tdTomatoflox/flox mice.

Induced constitutive expression of FGFR signalling in labeled AT2 progenitors was accomplished using a SftpcCreERT2/+; Rosa26rtTAflox/+; Tg(tet(o)caFgfr1/+; tdTomatoflox/+ mouse line (STOCK Sftpctm1(cre/ERT2,rtTA)HapGt(ROSA)26Sortm1.1(rtTA,EGFP)NagyTg(tetO-Fgfr3*R248C/Fgfr1)#Dor Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/sbel) [16]. Pregnant females carrying littermate experimental (expressing Tg(tet(o)caFgfr1) and control (without Tg(tet(o)caFgfr1) embryos were used for experiments.

In vivo activation of CreERT2/loxp and rtTA/tet(o) systems

Timed-pregnant females were used to conduct in vivo experiments, where the embryonic day (E) 0.5 was assumed to be noon on the day a vaginal copulation plug was found.

To activate the CreERT2/loxp system, tamoxifen, dissolved in corn oil, was administered at the desired timepoint via an intraperitoneal injection (Tam-IP) (Dosage: 0.1 mg tamoxifen/g mouse weight). To activate the rtTA/tet(o) system, doxycycline, dissolved either in PBS (for injection) or in water, was administered at the desired timepoint via an intraperitoneal injection (Dox-IP) (Dosage: 0.0015 mg doxycycline/g mouse weight), or through drinking water (Dosage: 200 µg doxycycline/ml water).

Euthanasia

At the endpoint of an experiment, a lethal dose of pentobarbital sodium was administered to animals via IP injection (Dosage: 0.4 mg pentobarbital/g mouse weight). After breathing ceased and a lack of pupil response to light was observed, cervical dislocation was performed to ensure death. Embryos were then harvested and briefly kept in PBS on ice before lung dissection.

Embryonic lung dissection and imaging

Shortly after embryo harvest, lungs were dissected under a stereomicroscope as detailed in Jones and Bellusci [17]. Briefly, embryos were placed in PBS in a petri dish. Tails were removed for genotyping. Working with fine-tipped forceps and dissecting scissors, lungs were very carefully and gently removed from the chest cavity to avoid any tissue damage. Lungs were positioned in the petri dish in PBS and were imaged. Whole lungs were used for FACS, left lobes were taken for RNA isolation, and right lobes were processed for paraffin embedding.

DNA isolation and PCR

DNA was isolated from embryonic tails and genotyped following standard lab procedures. PCR products were detected using capillary gel electrophoresis. A list of gene-specific primers can be found in supplementary Table S1.

RNA isolation and RT-qPCR

FACS-isolated AT1 and AT2 cells were put in 700 µl QIAzol Lysis Reagent (Qiagen), and lysed by vortexing. Total RNA was isolated using the miRNeasy Mini Kit (Qiagen), and eluted in 30 µl RNase-free water. RNA amount and purity were assessed using a NanoDrop 2000c spectrophotometer. Up to 1 µg of total RNA for each sample was reverse transcribed using the QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany).

Primers were designed to amplify specific mature mRNAs using NCBI’s primer-BLAST option (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) (last accessed, 07-10-2022). A list of gene-specific primers can be found in supplementary Table S2.

qPCR reaction mixtures were set up using the PowerUp SYBR Green Master Mix (Thermo Fisher, Schwerte, Germany), with a final volume of 20 µl for each reaction. Samples were run with two technical replicates on a LightCycler 480II using the following protocol: UDG activation at 50 °C for 2 min; DNA polymerase activation at 95 °C for 2 min; and 40 cycles of denaturation at 95 °C for 15 s, annealing at 60 °C for 15 s, and extension at 72 °C for 1 min. To validate amplification specificity, a dissociation step was also included for each sample. Threshold cycles (Ct) were calculated and used for relative expression analyses, using mouse Hprt as the reference gene.

∆Ct values were calculated according to the following formula:

$$\Delta C_}} C_}\;}}} - C_}\;}}} .$$

Unpaired two-tailed Student’s t tests were performed on the ∆Ct values, which can be assumed to be normally distributed. Number of ‘n’ and significance level is indicated either in the figures or in the figure legends.

Microarray

Differential gene expression was investigated using microarray as previously detailed [13, 18]. Briefly, for RNA concentrations greater than 50 ng/µl, the T7-protocol was followed. In this protocol, purified total RNA was amplified and Cy3-labeled using the LIRAK kit (Agilent Technologies) following the kit instructions. Per reaction, 200 ng of total RNA was used. The Cy3-labeled aRNA was hybridized overnight to 8 × 60 K 60mer oligonucleotide spotted microarray slides (Agilent Technologies, design ID 028005).

For experiments where samples yielded less than 50 ng/µl of RNA, the SPIA-protocol was utilized. In this protocol, purified total RNA was amplified using the Ovation PicoSL WTA System V2 kit (NuGEN Technologies). Per sample, 2 µg amplified cDNA was Cy-labeled using the SureTag DNA labeling kit (Agilent Technologies). The Cy3-labeled aRNA was hybridized overnight to 8 × 60K 60mer oligonucleotide spotted microarray slides (Agilent Technologies, design ID 074809).

For each protocol, hybridization, washing and drying of the slides followed the Agilent hybridization protocol. The dried slides were scanned at 2 µm/pixel resolution using the InnoScan is900 (Innopsys). Image analysis was performed with Mapix 6.5.0 software, and calculated values for all spots were saved as GenePix results files. Stored data were evaluated using the R software (version 3.3.2) (https://www.r-project.org/) and the limma package (version 3.30.13) from BioConductor (http://bioconductor.org/packages/release/bioc/html/limma.html). Gene annotation was supplemented by NCBI gene IDs via biomaRt (last accessed 31-03-2021).

FACS

The procedure to isolate RFP-labeled alveolar cells has been previously published in our lab [19]. In brief, after harvested lungs were dissociated in dispase and Collagenase Type IV at 37 °C for 40 min. with frequent agitation, single-cell suspensions were passed serially through 100-, 70- and 40-μm cell strainers (BD Biosciences). Red blood cells were eliminated using RBC lysis buffer (Sigma‐Aldrich), according to the manufacturer's protocol. Cells were then pelleted and resuspended in FACS buffer (0.1% sodium azide, 5% fetal calf serum (FCS), 0,05% in PBS) before being stained with antibodies: anti‐EpCAM (APC-Cy7‐conjugated, Biolegend,1:50), CD49F (APC‐conjugated, Biolegend,1:50), and anti‐PDPN (FITC‐conjugated, Biolegend, 1:20) for 20 min on ice in the dark, followed by washing. Next, cells were washed and stained with SYTOX (Invitrogen) according to the manufacturer’s instructions, to eliminate dead cells. Finally, flow cytometry and cell sorting were conducted using a FACSAria III cell sorter (BD Biosciences). Data were analyzed using FlowJo software version X (FlowJo, LLC).

Flow cytometry values were used in the figures. Significance was determined by unpaired two-tailed Student’s t tests. All data are presented as mean ± SEM. Values of p < 0.05 were considered significant. The number of independent samples (n) can be found in the figures.

Immunofluorescence

Freshly dissected E18.5 right lung lobes were washed briefly in sterile PBS, then fixed in 4% PFA for 4 h at 4 °C, and then washed again in PBS (3 × 5 min). Lungs were dehydrated by successive washes in a graded ethanol series (30, 50, 70, 100, 100%) for 5 min each, and then stored in 100% ethanol at − 20 °C until further processing.

For paraffin embedding, lungs were washed in Xylol (3 × 5 min, or until clear), incubated for 1 h at 60 °C in a 1:1 Xylol/paraffin mixture, washed in pure paraffin (3 × 20 min) at 60 °C, and then stored in pure paraffin overnight at 60 °C. Lungs were then embedded in paraffin blocks and sectioned using a microtome to a thickness of 3–5 µm. Sections were placed in a 40 °C water bath for approximately 30 min, and then placed on glass slides and incubated at 37 °C overnight.

Before antibody staining, sections were first washed with gentle shaking in Xylol (3 × 10 min), and then in serial dilutions of ethanol (100, 70, 50, and 30%) for 3 min each, and finally in distilled water for 5 min. Sections were then washed with PBST (1 × PBS + 0.1% TWEEN20) (3 × 5 min). Blocking solution (1 × PBS + 3% bovine serum albumin (BSA) + 5% goat serum (GS) + 0.4% TritonX) was then added atop each section for 1 h at room temperature. Primary antibodies were added to incubation buffer (1 × PBS + 1.5% BSA + 2.5% GS + 0.2% TritonX) and samples were incubated overnight at 4 °C (anti-Hopx (Atlas antibodies HPA030180), anti-Ki67 (Invitrogen 14-5698-82), and anti-PDPN (Invitrogen 14-5381-82) were added at 1:200 dilution and anti-proS-PC (Millipore AB3786) and anti-proS-PB (Abcam AB40876) at 1:500 dilution). Following primary antibody incubation, samples were washed in PBST (3 × 5 min) and secondary antibodies were added (all at a 1:500 dilution) for 1 h at room temperature, in the dark. Samples were washed in PBST (3 × 10 min) and PBS for 5 min, with gentle shaking. Finally, ProLong Gold antifade reagent with DAPI (Invitrogen) was added to each section and covered with a glass coverslip.

Proliferation and apoptosis

Proliferation was assessed using antibody staining against Ki67 (1:200 dilution). Due to the fact that Ki67 staining requires antigen retrieval and that antigen retrieval destroys the RFP protein, tomato RFP-positive cells were imaged just prior to the antigen retrieval step. After secondary staining, the original RFP images were used to identify the same region of the lung for re-imaging. Afterward, the two images of the region of interest were overlaid and cropped so that the corresponding cells matched perfectly. Images were then separated into individual channels and manually quantified using FIJI software.

Apoptosis was assessed on paraffin sections via the TdT-mediated dUTP Nick-End Labelling (TUNEL) assay using the DeadEnd Fluorometric TUNEL System (Promega) according to the manufacturer’s instructions. Apoptosis was not quantified because the number of apoptotic cells in each sample were too few.

Correlating FGFR2b signatures to published scRNA-seq data

E17.5 lung scRNA-seq expression data from Frank et al. [7] was accessed (GEO GSE113320) and the AT1 and AT2 lineages were clustered and displayed on a tSNE plot using the Seurat v2.2 R package pipeline (http://satijalab.org/seurat/), similar to the original paper [7]. FGFR2b gene signature scores were calculated as previously described [20]. Subclustering of cluster 4 (mature AT2s) was performed using the updated Seurat v3. Plots were displayed using UMAP to increase clustering resolution. SCTransform from the Seurat package was used for data normalization and scaling. PCA was followed by UMAP using pc = 30. Clusters were identified using the FindClusters function, with a resolution of 0.5. For the heatmap, the top 50 genes of each subcluster were selected based on the average log10fold changes, and scaled, centered and normalized expression was displayed. The threshold to identify differentially expressed genes was set to log10fold change = 0.25.

Correlating FGFR2b signatures to published scRNA-seq data from a bleomycin-induced lung injury study

For this analysis, the pre-processed data set published in Strunz et al. [21] was retrieved from the listed GitHub repository (https://github.com/theislab/2019_Strunz) and explored with SCANPY (v1.6.0), as previously described [22]. This study assessed the gene expression changes of murine lungs after bleomycin injury at multiple time points with Drop-seq. The whole lung data set was further used without modification, whereas the EpCAMpos enriched data set with densely sampled time points (days 1–14, 21, 28, 35, 56) was subset to cell types belonging to the alveolar epithelium (AT1, AT2, Krt8 ADI, activated AT2). The principal components and knn graph (n_pcs = 10, n_neighbors = 20) were re-calculated and formed the input for the UMAP algorithm.

To quantify the enrichment of the FGFR2b signatures, a score was calculated using SCANPY’s tl.score_genes() on both the whole lung data set and the alveolar epithelium subset. The input signatures consisted of 42 genes for time point E12.5, 76 genes for E14.5 and 48 genes for E16.5. For visual inspection, the three scores were then overlaid onto the UMAP embeddings for both data sets.

In situ hybridization expression data

To assess the expression patterns of genes in embryonic lungs, the online database ‘Genepaint’, which contains in situ hybridization data for many genes expressed in E14.5 lungs, was used (https://gp3.mpg.de/) (last accessed 01-03-2022). Each of the genes significantly downregulated in our E16.5 + 9 h experiment was assessed, and the ones which were clearly present were chosen for the figure.

Microarray analyses

As detailed elsewhere [13, 18], mean spot signals were background corrected with an offset of 1 using the NormExp procedure on the negative control spots. The logarithms of the background-corrected values were quantile-normalized. The normalized values were then averaged for replicate spots per array. From different probes addressing the same NCBI gene ID, the probe showing the maximum average signal intensity over the samples was used in subsequent analyses. Genes were ranked for differential expression using an unpaired two-tailed Student’s t test on a moderated t statistic, and heatmaps were generated displaying genes according to descending p values. Gene set tests were done on the ranks of the t values, using the function ‘geneSetTest’ in the limma package from BioConductor. The number of independent samples (n) can be found in the figures. Gene sets were either user defined or, for pathway analyses, according to the KEGG database (last accessed 31-03-2021).

The data from the microarray experiments have been deposited in the NCBI's gene expression omnibus (GEO accession GSE115880).

Manual quantification of immunostained cells

Immunostained sections from each sample (n = 3) were imaged at 63×. Multiple images (between 7 and 15) of distal alveolar regions were captured per section and exported as TIFF files. Images were processed and analyzed using FIJI (version 2.1.0/1.53c) [23]. First, images were separated into their component colour channels (red, green and blue). The average background value of each channel was determined and subtracted from the total signal value. Separately, red-positive and green-positive were manually labelled and counted. These fields were then overlaid and the double-positive cells were tallied.

Average cell counts were determined per sample. Unpaired two-tailed Student’s t tests were performed on the average values. All data are presented as mean ± SEM. Values of p < 0.05 were considered significant. The number of independent samples (n) can be found in the figures and figure legends.

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