FGF Signaling Pathway in the Developing Chick Lung: Expression and Inhibition Studies

Abstract

BackgroundFibroblast growth factors (FGF) are essential key players during embryonic development. Through their specific cognate receptors (FGFR) they activate intracellular cascades, finely regulated by modulators such as Sprouty. Several FGF ligands (FGF1, 2, 7, 9, 10 and 18) signaling through the four known FGFRs, have been implicated in lung morphogenesis. Although much is known about mammalian lung, so far, the avian model has not been explored for lung studies.Methodology/Principal FindingsIn this study we provide the first description of fgf10, fgfr1-4 and spry2 expression patterns in early stages of chick lung development by in situ hybridization and observe that they are expressed similarly to their mammalian counterparts. Furthermore, aiming to determine a role for FGF signaling in chick lung development, in vitro FGFR inhibition studies were performed. Lung explants treated with an FGF receptor antagonist (SU5402) presented an impairment of secondary branch formation after 48 h of culture; moreover, abnormal lung growth with a cystic appearance of secondary bronchi and reduction of the mesenchymal tissue was observed. Branching and morphometric analysis of lung explants confirmed that FGFR inhibition impaired branching morphogenesis and induced a significant reduction of the mesenchyme.Conclusions/SignificanceThis work demonstrates that FGFRs are essential for the epithelial-mesenchymal interactions that determine epithelial branching and mesenchymal growth and validate the avian embryo as a good model for pulmonary studies, namely to explore the FGF pathway as a therapeutic target.

Three-Dimensional Culture and FGF Signaling Drive Differentiation of Murine Pluripotent Cells to Distal Lung Epithelial Cells

“…This finding is consistent with our results, as signaling via FGFR2c (and/or FGFR1c) seems to be critical for the initial induction of NKX2-1 expression, whereas FGFR2b appears to play a role in organization, and further proliferation and differentiation. FGFR1c, the functionally active isoform of FGFR1 [51], is expressed throughout the epithelium and mesenchyme of the early chick lung [52], in agreement with our Fgfr1c expression findings in E11.5 EPCAM + (epithelium) and EPCAM -(mesenchymal) cells. Fgfr1-null mice have demonstrated essential functions in embryonic development prior to early lung formation [51,53].…”

Apical constriction initiates new bud formation during monopodial branching of the embryonic chicken lung

“…We found little discernible apoptosis within the airway epithelium at these stages (supplementary material Fig. S5), consistent with previous reports (Moura et al, 2011). We then included cell proliferation in our computational model to test whether the combination of proliferation and apical constriction could generate the more fully developed bud morphology observed in experiments (see b1 in Fig.…”

“…2H,I). Branching morphogenesis of the avian (Moura et al, 2011) and mammalian (Bellusci et al, 1997;Abler et al, 2009) lungs is regulated via signaling through FGF, which has also been shown to induce actomyosin contraction during morphogenesis of the inner ear (Sai and Ladher, 2008) and apical constriction of the rosettes that form the lateral line in zebrafish (Harding and Nechiporuk, 2012). Consistently, we found that treatment with an FGF receptor kinase inhibitor, SU5402 (5 µM), blocked apical constriction (supplementary material Fig.…”

“…Similar to lung development in mice (Bellusci et al, 1997;Park et al, 1998), FGF10 is expressed focally in the embryonic chicken lung in regions of mesenchyme adjacent to emerging buds (Sakiyama et al, 2003;Moura et al, 2011). A feedback mechanism between FGF10 and the transcription factors Tbx4 in the mesenchyme and Nkx2.1 in the epithelium has been shown to regulate monopodial branching in the developing chicken lung (Sakiyama et al, 2003).…”

A photoactivatable small-molecule inhibitor for light-controlled spatiotemporal regulation of Rho kinase in live embryos

“…Their ease of delivery, reversibility, concentration-dependent phenotypes and cross-species activity have made these compounds invaluable for investigating the role of specific signaling pathways in dynamic morphogenetic processes including organogenesis and regeneration (e.g. Mathew et al, 2009;Moura et al, 2011). Although small molecule exposure can be limited to precise developmental windows, the effects are not spatially restricted, limiting their utility for studies of a specific region of interest within the context of the whole embryo.…”