Characterization of Tg(Etv4-GFP) and Etv5RFP Reporter Lines in the Context of Fibroblast Growth Factor 10 Signaling During Mouse Embryonic Lung Development

Members of the PEA3 transcription factors are emerging as bone fide targets for fibroblast growth factor (FGF) signaling. Among them, ETV4 and ETV5 appear to mediate FGF10 signaling during early embryonic lung development. In this paper, recently obtained Tg(Etv4-GFP) and Etv5CreERT2−RFP fluorescent reporter lines were generally characterized during early embryonic development and in the context of FGF10 signaling, in particular. We found that both Tg(Etv4-GFP) and Etv5CreERT2−RFP were primarily expressed in the epithelium of the lung during embryonic development. However, the expression of Etv5CreERT2−RFP was much higher than that of Tg(Etv4-GFP), and continued to increase during development, whereas Tg(Etv4-GFP) decreased. The expression patterns of the surrogate fluorescent protein GFP and RFP for ETV4 and ETV5, respectively, agreed with known regions of FGF10 signaling in various developing organs, including the lung, where ETV4-GFP was seen primarily in the distal epithelium and to a lesser extent in the surrounding mesenchyme. As expected, ETV5-RFP was restricted to the lung epithelium, showing a decreasing expression pattern from distal buds to proximal conducting airways. FGF10 inhibition experiments confirmed that both Etv4 and Etv5 are downstream of FGF10 signaling. Finally, we also validated that both fluorescent reporters responded to FGF10 inhibition in vitro. In conclusion, these two reporter lines appear to be promising tools to monitor FGF10/FGFR2b signaling in early lung development. These tools will have to be further validated at later stages and in other organs of interest

A Comprehensive Analysis of Fibroblast Growth Factor Receptor 2b Signaling on Epithelial Tip Progenitor Cells During Early Mouse Lung Branching Morphogenesis

This study demonstrates that FGF10/FGFR2b signaling on distal epithelial progenitor cells, via ß-catenin/EP300, controls, through a comprehensive set of developmental genes, morphogenesis, and differentiation. Fibroblast growth factor (FGF) 10 signaling through FGF receptor 2b (FGFR2b) is mandatory during early lung development as the deletion of either the ligand or the receptor leads to lung agenesis. However, this drastic phenotype previously hampered characterization of the primary biological activities, immediate downstream targets and mechanisms of action. Through the use of a dominant negative transgenic mouse model (Rosa26rtTA; tet(o)sFgfr2b), we conditionally inhibited FGF10 signaling in vivo in E12.5 embryonic lungs via doxycycline IP injection to pregnant females, and in vitro by culturing control and experimental lungs with doxycycline. The impact on branching morphogenesis 9 h after doxycycline administration was analyzed by morphometry, fluorescence and electron microscopy. Gene arrays at 6 and 9 h following doxycycline administration were carried out. The relationship between FGF10 and ß-catenin signaling was also analyzed through in vitro experiments using IQ1, a pharmacological inhibitor of ß-catenin/EP300 transcriptional activity. Loss of FGF10 signaling did not impact proliferation or survival, but affected both adherens junctions (up-regulation of E-cadherin), and basement membrane organization (increased laminin). Gene arrays identified multiple direct targets of FGF10, including main transcription factors. Immunofluorescence showed a down-regulation of the distal epithelial marker SOX9 and mis-expression distally of the proximal marker SOX2. Staining for the transcriptionally-active form of ß-catenin showed a reduction in experimental vs. control lungs. In vitro experiments using IQ1 phenocopied the impacts of blocking FGF10. This study demonstrates that FGF10/FGFR2b signaling on distal epithelial progenitor cells via ß-catenin/EP300 controls, through a comprehensive set of developmental genes, cell adhesion, and differentiation

Evidence for a lipofibroblast to collagen triple helix repeat containing 1positive myofibroblast reversible switch during the development and resolution of lung fibrosis

Question addressed in the study: Genetic lineage tracing of alpha smooth muscle actin-positive (Acta2+) cells in the context of bleomycin-induced pulmonary fibrosis in mice has shown that activated myofibroblasts (aMYFs) differentiate into lipofibroblasts (LIFs) during fibrosis resolution. However, the heterogeneity of the different mesenchymal cell subclusters during fibrosis formation and their contribution to the aMYFs as well as the fate of aMYFs during fibrosis resolution are still unclear. Materials and methods: Two-month-old Tg(Acta2-CreERT2); tdTomatoflox mice were used to label Acta2pos cells before (Tam-Bleo condition) or after (Bleo-Tam condition) bleomycin administration. Using scRNA-seq, the origin and fate of Acta2pos cells converging on the aMYF/Cthrc1pos myofibroblast lineage, were analyzed at the peak of fibrosis formation (day 14) as well as during resolution (day 60). Mining of human IPF scRNA-seq data was also carried out. The function of resident mesenchymal cells derived from saline or bleomycin-injured lungs during fibrosis formation or resolution in supporting AT2 progenitor cell renewal was assessed using alveolar organoid assays. Results: By employing dimensionality reduction and UMAP visualization, aMYF/Cthrc1pos cells were identified and characterized in three scRNA-seq conditions: Saline, Tam-Bleo and Bleo-Tam. Acta2pos cells isolated from saline-treated mice revealed the presence of Acta2pos cells in the expected airway and vascular smooth muscle cells, but also in alveolar fibroblasts, peribronchial and adventitial fibroblasts as well as in a restricted number of Cthrc1pos cells indicating a primed pro-fibrotic population in non-injured lung. In Tam-Bleo lungs, minimal contribution of these cells to the Cthrc1pos pool was observed. By contrast, labeling of Acta2pos cells following bleo administration (Bleo-Tam) led to massive labeling of Cthrc1pos cells, which following fine clustering were grouped into 4 subclusters named Ct1-4. Interestingly, cells in cluster Ct1 were identified as Cthrc1low and displayed a strong LIF signature (LIFhigh) while cells in cluster Ct2 were LIFlow and Cthrc1high. Examination of Ct1 and Ct2 subclusters during fibrosis resolution suggested that Ct2 transition back to an Ct1 status and eventually differentiate into Cthrc1low LIFhigh alveolar fibroblasts. Analysis of the alveolar fibroblast cluster revealed the presence of three subclusters (Al1-3). Cluster Al3 appeared only in Bleo-Tam and displayed a fibrotic signature (Sfrp1, Eln, Ltbp2, Spp1high). These cells, which originate from Acta2neg alveolar fibroblasts are suggested to differentiate into the Cthrc1low LIFhigh Ct1 subcluster. Data mining of human scRNA-seq from fibrotic lungs supported the conservation of the heterogeneity of the CTHRC1pos population. Alveolosphere assays to test the activity of the resident mesenchymal cell niche indicated that the alveolar fibroblast did not recover their supportive function for the proliferation of the alveolar epithelial type 2 stem cells following fibrosis resolution. Answer to the question: Our results support a LIF-to-aMYF reversible switch during fibrosis formation and resolution

Insights into the Black Box of Intra-Amniotic Infection and Its Impact on the Premature Lung: From Clinical and Preclinical Perspectives

Intra-amniotic infection (IAI) is one major driver for preterm birth and has been demonstrated by clinical studies to exert both beneficial and injurious effects on the premature lung, possibly due to heterogeneity in the microbial type, timing, and severity of IAI. Due to the inaccessibility of the intra-amniotic cavity during pregnancies, preclinical animal models investigating pulmonary consequences of IAI are indispensable to elucidate the pathogenesis of bronchopulmonary dysplasia (BPD). It is postulated that on one hand imbalanced inflammation, orchestrated by lung immune cells such as macrophages, may impact on airway epithelium, vascular endothelium, and interstitial mesenchyme, resulting in abnormal lung development. On the other hand, excessive suppression of inflammation may as well cause pulmonary injury and a certain degree of inflammation is beneficial. So far, effective strategies to prevent and treat BPD are scarce. Therapeutic options targeting single mediators in signaling cascades and mesenchymal stromal cells (MSCs)-based therapies with global regulatory capacities have demonstrated efficacy in preclinical animal models and warrant further validation in patient populations. Ante-, peri- and postnatal exposome analysis and therapeutic investigations using multiple omics will fundamentally dissect the black box of IAI and its effect on the premature lung, contributing to precisely tailored and individualized therapies

Characterization of Tg(Etv4-GFP) and Etv5RFP Reporter Lines in the Context of Fibroblast Growth Factor 10 Signaling During Mouse Embryonic Lung Development

International audienceMembers of the PEA3 transcription factors are emerging as bone fide targets for fibroblast growth factor (FGF) signaling. Among them, ETV4 and ETV5 appear to mediate FGF10 signaling during early embryonic lung development. In this paper, recently obtained Tg(Etv4-GFP) and Etv5 CreERT2−RFP fluorescent reporter lines were generally characterized during early embryonic development and in the context of FGF10 signaling, in particular. We found that both Tg(Etv4-GFP) and Etv5 CreERT2−RFP were primarily expressed in the epithelium of the lung during embryonic development. However, the expression of Etv5 CreERT2−RFP was much higher than that of Tg(Etv4-GFP), and continued to increase during development, whereas Tg(Etv4-GFP) decreased. The expression patterns of the surrogate fluorescent protein GFP and RFP for ETV4 and ETV5, respectively, agreed with known regions of FGF10 signaling in various developing organs, including the lung, where ETV4-GFP was seen primarily in the distal epithelium and to a lesser extent in the surrounding mesenchyme. As expected, ETV5-RFP was restricted to the lung epithelium, showing a decreasing expression pattern from distal buds to proximal conducting airways. FGF10 inhibition experiments confirmed that both Etv4 and Etv5 are downstream of FGF10 signaling. Finally, we also validated that both fluorescent reporters responded to FGF10 inhibition in vitro. In conclusion, these two reporter lines appear to be promising tools to monitor FGF10/FGFR2b signaling in early lung development. These tools will have to be further validated at later stages and in other organs of interest

Fgfr2b signaling is essential for the maintenance of the alveolar epithelial type 2 lineage during lung homeostasis in mice

Deutsche Forschungsgemeinschaft (DFG); ROR-ID:018mejw6

Highlighting fibroblast plasticity in lung fibrosis: the WI-38 cell line as a model for investigating the myofibroblast and lipofibroblast switch

Background: Myofibroblasts (MYFs) are generally considered the principal culprits in excessive extracellular matrix deposition and scar formation in the pathogenesis of lung fibrosis. Lipofibroblasts (LIFs), on the other hand, are defined by their lipid-storing capacity and are predominantly found in the alveolar regions of the lung. They have been proposed to play a protective role in lung fibrosis. We previously reported that a LIF to MYF reversible differentiation switch occurred during fibrosis formation and resolution. In this study, we tested whether WI-38 cells, a human embryonic lung fibroblast cell line, could be used to study fibroblast differentiation towards the LIF or MYF phenotype and whether this could be relevant for idiopathic pulmonary fibrosis (IPF). Methods: Using WI-38 cells, Fibroblast (FIB) to MYF differentiation was triggered using TGF-β1 treatment and FIB to LIF differentiation using Metformin treatment. We also analyzed the MYF to LIF and LIF to MYF differentiation by pre-treating the WI-38 cells with TGF-β1 or Metformin respectively. We used IF, qPCR and bulk RNA-Seq to analyze the phenotypic and transcriptomic changes in the cells. We correlated our in vitro transcriptome data from WI-38 cells (obtained via bulk RNA sequencing) with the transcriptomic signature of LIFs and MYFs derived from the IPF cell atlas as well as with our own single-cell transcriptomic data from IPF patients-derived lung fibroblasts (LF-IPF) cultured in vitro. We also carried out alveolosphere assays to evaluate the ability of the proposed LIF and MYF cells to support the growth of alveolar epithelial type 2 cells. Results: WI-38 cells and LF-IPF display similar phenotypical and gene expression responses to TGF-β1 and Metformin treatment. Bulk RNA-Seq analysis of WI-38 cells and LF-IPF treated with TGF-β1, or Metformin indicate similar transcriptomic changes. We also show the partial conservation of the LIF and MYF signature extracted from the Habermann et al. scRNA-seq dataset in WI-38 cells treated with Metformin or TGF-β1, respectively. Alveolosphere assays indicate that LIFs enhance organoid growth, while MYFs inhibit organoid growth. Finally, we provide evidence supporting the MYF to LIF and LIF to MYF reversible switch using WI-38 cells. Conclusions: WI-38 cells represent a versatile and reliable model to study the intricate dynamics of fibroblast differentiation towards the MYF or LIF phenotype associated with lung fibrosis formation and resolution, providing valuable insights to drive future research.Deutsche Forschungsgemeinschaft (DFG); ROR-ID:018mejw6