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

Abstract

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.

Fgf10 Signaling-Based Evidence for the Existence of an Embryonic Stage Distinct From the Pseudoglandular Stage During Mouse Lung Development

“…As the main Fgfr2b ligand expressed in the lung at this early developmental stage (E9.5-E12.5) is Fgf10 (Bellusci et al, 1997;Jones et al, 2019), our experimental approach leads to the inhibition of Fgf10 activity. We will therefore refer throughout this study to Fgf10 inhibition after doxycycline exposure.…”

“…These samples, for each time point and condition, were pooled for the transcriptomic analysis. Figure 4F uses, as a base for the analysis, the genes previously identified to be downstream of Fgf10 signaling at E12.5 in the mouse lung (Jones et al, 2019). This signature corresponds to a stage where in mouse, Fgf10's role is mostly dedicated to the branching process.…”

“…This signature corresponds to a stage where in mouse, Fgf10's role is mostly dedicated to the branching process. We analyzed the expression of these genes in the hiPSC treated vs. not treated with FGF10 for 12 h and 24 h. The rationale for this analysis is that the genes found to be part of the Fgf10 transcriptomic signature (which display downregulated expression in the comparison between soluble Fgfr2b and control lungs, see Jones et al, 2019 for details), should be upregulated in our gain of function experiment at least at one of the time points considered. Out of the 33 genes considered, only 11 were consistently conserved (these are written in red in Figure 4F and display upregulation at least at one of the times point eventually with no change at the other time point.…”

“…To test this hypothesis, we used a genetically modified mouse line which allows the inducible, robust and ubiquitous expression of a soluble form of Fgfr2b which will inhibit Fgf10 ligand activity at the protein level. This line has been extensively used and validated in mouse to study the role of Fgfr2 signaling in multiple organs (Parsa et al, 2010;Danopoulos et al, 2013;Jones et al, 2019Jones et al, , 2020. In this study, we transiently inhibited endogenous Fgf10 activity in mutant embryos at E9, E9.5, and E11 following intra-peritoneal exposure of the embryos to doxycycline and examined the resulting lung phenotype at E18.5.…”

3D cell culture models demonstrate a role for FGF and WNT signaling in regulation of lung epithelial cell fate and morphogenesis

“…Fgf10 gain-of-function prevents differentiation of epithelial tip cells toward the bronchial progenitor lineage and disrupts lung morphogenesis (Nyeng et al, 2008; Volckaert et al, 2013). Furthermore, FGF1, FGF2, FGF7, and FGF9, were found in fetal rodent lung, too (Han et al, 1992; Cardoso et al, 1997; Powell et al, 1998; Colvin et al, 2001; Jones et al, 2019). FGF7 acts as a proliferative factor for lung epithelium during lung development (Lebeche et al, 1999) and together with FGF2 it induces expression of surfactant proteins (Matsui et al, 1999).…”