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.

The specification, characterization, and fate of alveolar type 1 and type 2 (AT1 and AT2) progenitors during embryonic lung development are poorly defined. Current models of distal epithelial lineage formation fail to capture the heterogeneity and dynamic contribution of progenitor pools present during early development. Furthermore, few studies explore the pathways involved in alveolar progenitor specification and fate. In this paper, we build upon our previously published work on the regulation of airway epithelial progenitors by fibroblast growth factor receptor 2b (FGFR2b) signalling during early (E12.5) and mid (E14.5) pseudoglandular stage lung development. Our results suggest that a significant proportion of AT2 and AT1 progenitors are lineage-flexible during late pseudoglandular stage development, and that lineage commitment is regulated in part by FGFR2b signalling. We have characterized a set of direct FGFR2b targets at E16.5 which are likely involved in alveolar lineage formation. These signature genes converge on a subpopulation of AT2 cells later in development and are downregulated in AT2 cells transitioning to the AT1 lineage during repair after injury in adults. Our findings highlight the extensive heterogeneity of pneumocytes by elucidating the role of FGFR2b signalling in these cells during early airway epithelial lineage formation, as well as during repair after injury.