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

Matthew R Jones,Gianni Carraro,Arun Lingampally,Denise Al Alam,Cho-Ming Chao,Chengshui Chen,Jochen Wilhelm,Salma Dilai,Jin San Zhang,Saverio Bellusci,Parviz Minoo,Regina Mukhametshina,Eveline Baumgart-Vogt,Soula Danopoulos

doi:10.3389/fgene.2018.00746

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.

Highlights

In mice, the first morphological evidence of lung development is seen at embryonic day (E) 9.5 with the budding of the ventral foregut endoderm, forming the tracheal primordium ventrally and the esophagus dorsally
Given that wingless/integrase 1 (WNT)/ß-catenin signaling is vital for proper branching morphogenesis and cellular differentiation and that we observed a drastic reduction in activated ß-catenin in our experimental lungs, we investigated the degree to which the effect of inhibiting fibroblast growth factor 10 (FGF10) was mediated through WNT/ßcatenin signaling
We report the impacts on the epithelial tip cells of E12.5 lungs by blocking FGFR2b ligands, primarily FGF10

Summary

INTRODUCTION

The first morphological evidence of lung development is seen at embryonic day (E) 9.5 with the budding of the ventral foregut endoderm, forming the tracheal primordium ventrally and the esophagus dorsally. Fibroblast growth factor 10 (FGF10), signaling via its epithelial receptor FGFR2b, is sufficient to induce branching morphogenesis of isolated lung endoderm grown in Matrigel (Bellusci et al, 1997b). Almost twenty years after the discovery of FGF10 as a key growth factor regulating branching morphogenesis, the primary targets and biological activities controlled by FGF10 are still unclear (El Agha and Bellusci, 2014) Addressing this issue has been difficult, since loss of Fgf leads to lung agenesis, leaving little tissue to study; and while conditional deletions are possible, the lapse of time separating either constitutive or inducible Cre activity (in the case of a CreERT2 system) from complete gene inactivation is usually 24 to 48 h, it is difficult to distinguish between primary and secondary effects (Abler et al, 2009). Such knowledge will be instrumental to better understanding the role of FGF10 signaling at later stages of lung development, as well as during the repair process after injury

RESULTS

Discussion

MATERIALS AND METHODS

Method Details