Characterization of the role of fibroblast growth factor 10 (Fgf10) and its receptor Fgfr2b on multipotent epithelial progenitor cells during early lung development

Abstract

FGF10 signaling through FGFR2b is mandatory during early lung development as the deletion of either the ligand or the receptor leads to lung agenesis. In this dissertation, we report the role of FGF10/FGFR2b signaling on the early lung development (E12.5) by attenuating FGFR2b ligands primarily Fgf10 (loss of function), and by over-expressing Fgf10 (gain of function). In the loss of function part, we used a dominant negative transgenic mouse model (Rosa26rtTA; tg(tet(o)sFgfr2b)/+), we conditionally inhibited FGF10 signaling in vivo at E12.5 embryonic lungs via doxycycline IP injection to pregnant females, and in vitro by culturing control and experimental lungs with doxycycline. Both in vivo and in vitro experiments inhibiting Fgf10 resulted in arrested epithelial branching collapsed distal bud and dilatation of the mesenchyme; the impact on branching morphogenesis was also analyzed by morphometry, and electron microscopy (Jones, Dilai et al., 2019a). Gene arrays at 6 and 9 hours post dox IP was carried out and revealed the transcriptomic regulation of FGFR2b signaling with significant changes in cytokine-cytokine receptor interactions, calcium signaling, WNT signaling and Hedgehog signaling. By gene array analysis, we identified an FGF10 gene signature primarily composed of genes enriched in the epithelium and positively regulated by Fgf10. We also identified a set of lung specific transcription factors significantly regulated by Fgf10. The impact of blocking FGFR2b ligands signaling on the differentiation of multipotent epithelial progenitors was also identified, which demonstrate the loss of distal differentiation markers 9 hours after FGF10 inhibition and the proximalization of the tip epithelium which is enriched with differentiated genes encoding for AECII that are controlled by Fgf10. Furthermore, we assessed the well-established FGF10-SHH regulatory feedback loop during lung development as a means of validating our array (Figure 18E). Not only does our array detect the down-regulation of Shh within 6 hours of FGF10 inhibition, but also it picks up the expected delayed impacts on the downstream targets of Shh at 9 hours, including the up-regulation of Fgf10. Additionally, our array supports the reported data showing that the inactivation of FGF signaling regulated Etv4 and Etv5 in the multipotent epithelial progenitor cells during lung development and leads to the loss of Shh expression (Herriges et al., 2015). We therefore propose that FGF10 acts via FGFR2b, positively regulating the expression of Etv4/Etv5, which in turn transcriptionally regulates Shh expression, and therefore the downstream genes involved in the SHH pathway. In the gain of function part, over-expressing Fgf10 resulted in a lung more ramified compared to the control, increased epithelial surface, elongated branches, and dilated mesenchyme. Gene arrays analysis at 9 hours of Fgf10 over-expression revealed a regulation of FGFR2b signaling with significant alterations in cytokine-cytokine receptor interactions, cell adhesion molecules and ECM-receptor interaction, these data support changes in the rearrangements of the epithelial changes linked to impaired cell- cell or cell-matrix interactions. By the Comparison of the LoF and The GoF, we found a cluster containing genes that are down-regulated upon Fgf10 attenuation, and up-regulated upon Fgf10 over-expression these genes might be the one controlled by Fgf10. In summary, we validated the reporter mouse line Tg(Etv4/GFP) which appear to be a promising tool to monitor FGF10/FGFR2b signaling in early lung development. We also carried out a comprehensive analysis of fibroblast growth factor receptor 2b signaling on multipotent epithelial progenitor cells during early mouse lung branching morphogenesis, where we defined the FGF10 transcriptomic signature which will be instrumental to design new mechanistic studies concerning the role of FGF10 in alveolar epithelium formation during development, as well as maintenance during homeostasis and repair after injury.