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.

Branching morphogenesis is the basic developmental mode common to organs such as the lungs that undergo a process of ramification from a rudimentary tree. However, the precise molecular and cellular bases underlying the formation of branching organs are still unclear. As inactivation of fibroblast growth factor receptor 2b (Fgfr2b) signaling during early development leads to lung agenesis, thereby preventing the analysis of this pathway at later developmental stages, we used transgenic mice to induce expression of a soluble form of Fgfr2b to inactivate Fgfr2b ligands at embryonic day (E) 14.5, corresponding to the mid-pseudoglandular stage of lung development. We identified an Fgfr2b signaling signature comprised of 46 genes enriched in the epithelium, some of which were common to, but most of them distinct from, the previously identified Fgfr2b signaling signature at E12.5. Our results indicate that Fgfr2b signaling at E14.5 controls mostly proliferation and alveolar type 2 cell (AT2) differentiation. In addition, inhibition of Fgfr2b signaling at E14.5 leads to morphological and cellular impairment at E18.5, with defective alveolar lineage formation. Further studies will have to be conducted to elucidate the role of Fgfr2b signaling at successive stages (canalicular/saccular/alveolar) of lung development as well as during homeostasis and regeneration and repair after injury.

Idiopathic Pulmonary Fibrosis (IPF) is an end-stage lung disease characterized by excessive extracellular matrix (ECM) deposition from activated myofibroblasts (MYFs) and tissue scarring. Eventually leading to stiffening of the lung, capable of assuming only limited gas exchange function. So far two drugs, pirfenidone [acting via TGF-β (transforming growth factor beta) inhibition] and nintedanib (a pan-tyrosine kinase receptor inhibitor) have been approved for IPF patients. They both act on the activated MYF by reducing the expression of fibrotic markers. Unfortunately, these drugs are only slowing down fibrosis formation and as such do not represent a cure for this lethal, devastating disease. We previously reported that activated MYF originate, at least in part, from lung fibroblast resident cells called lipofibroblasts (LIF). During resolution, these activated MYF can transdifferentiate into LIF. We propose that this reversible myogenic/lipogenic transdifferentiation switch paradigm can be used to screen for drugs capable of triggering the lipogenic differentiation of activated MYFs. Ideally, these drugs should also induce the reduction of pro-fibrotic markers alpha smooth muscle actin2 (ACTA2) and collagen 1A1 (COL1A1) in activated MYF and as such would represent important alternatives to the approved drugs. The goal of this review is to summarize the current knowledge and limitations of the current strategies aiming to carry out methodical pre-clinical drug screening in pertinent in vitro, ex vivo, and in vivo models of IPF. These models include (1) in vitro culture of primary fibroblasts from IPF patients, (2) ex vivo culture of precision cut lung slices from end-stage IPF lungs obtained from transplant patients, and (3) bleomycin-induced fibrosis mouse models in the context of lineage tracing of activated MYF during resolution. For all these assays, we propose the innovative use of lipogenic read outs for the LIFs.

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 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.