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

Background: Fibroblast 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 Findings: In 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/Significance: This work demonstrates that FGFRs are essential for the epithelial-mesenchymal interactions tha

Canonical wnt signaling activity in early stages of chick lung development

Wnt signaling pathway is an essential player during vertebrate embryonic development which has been associated with several developmental processes such as gastrulation, body axis formation and morphogenesis of numerous organs, namely the lung. Wnt proteins act through specific transmembrane receptors, which activate intracellular pathways that regulate cellular processes such as cell proliferation, differentiation and death. Morphogenesis of the fetal lung depends on epithelial-mesenchymal interactions that are governed by several growth and transcription factors that regulate cell proliferation, fate, migration and differentiation. This process is controlled by different signaling pathways such as FGF, Shh and Wnt among others. Wnt signaling is recognized as a key molecular player in mammalian pulmonary development but little is known about its function in avian lung development. The present work characterizes, for the first time, the expression pattern of several Wnt signaling members, such as wnt-1, wnt-2b, wnt-3a, wnt-5a, wnt-7b, wnt-8b, wnt-9a, lrp5, lrp6, sfrp1, dkk1, β-catenin and axin2 at early stages of chick lung development. In general, their expression is similar to their mammalian counterparts. By assessing protein expression levels of active/total β-catenin and phospho-LRP6/LRP6 it is revealed that canonical Wnt signaling is active in this embryonic tissue. In vitro inhibition studies were performed in order to evaluate the function of Wnt signaling pathway in lung branching. Lung explants treated with canonical Wnt signaling inhibitors (FH535 and PK115-584) presented an impairment of secondary branch formation after 48 h of culture along with a decrease in axin2 expression levels. Branching analysis confirmed this inhibition. Wnt-FGF crosstalk assessment revealed that this interaction is preserved in the chick lung. This study demonstrates that Wnt signaling is crucial for precise chick lung branching and further supports the avian lung as a good model for branching studies since it recapitulates early mammalian pulmonary development.Rute S. Moura was supported by a grant of ON.2 SR&TD Integrated Program (N-01-01-0124-01-07), ref: UMINHO/BPD/31/2013. The funders had no role in study design, data collection and analysis

<i>In vitro</i> Wnt signaling inhibition and <i>spry2</i> expression.

<p>Representative examples of stage b2 lung explant culture, at D0∶0h (A, D, G, J) and D2∶48h (B, E, H, K) treated with DMSO (A, B), 20 µM (D, E), 30 µM (G, H) and 40 µM FH535 (J, K) and probed with <i>spry2</i> (C, F, I, L); n = 3 for each stage. Magnification: A, B, D, E, G, H, J, K –4x; C, F, I, L –5x. The signal observed in the most proximal region of the lung is due to the accumulation of developing solution.</p

<i>In vitro</i> Wnt signaling inhibition (FH535) and branching analysis of lung explants.

<p>Representative examples of stage b2 lung explant culture, at D0∶0h (<b>A, D, G, J</b>) and D2∶48h (<b>B, E, H, K</b>) treated with DMSO (A, B), 20 µM (D, E), 30 µM (G, H) and 40 µM FH535 (J, K) and probed with <i>axin2</i> (<b>C, F, I, L</b>); n = 5 for each stage. Magnification: A, B, D, E, G, H, J, K –4x; C, F, I, L –5x. <b>M</b>: Branching analysis of stage b1 (n≥15 for each condition), b2 (n≥40 for each condition) and b3 (n≥30 for each condition) explants treated with DMSO and FH535 (20, 30 and 40 µM). Results are expressed as D2/D0 ratio. Data is represented as mean ± SEM. p<0.001 * <i>vs</i> DMSO, § <i>vs</i> 20 µM of FH535, ¥ <i>vs</i> 30 µM of FH535.</p

Activity of Wnt/β-catenin pathway in the embryonic chick lung.

<p>(A) Western blot analysis of active and total β-catenin in stage b1, b2 and b3 lungs, and stage 24 limb (as positive control). Control loading was performed using β-tubulin (55 kDa). Total and active β-catenin correspond to 92 kDa. (B) Semi-quantitative analysis for active and total β-catenin. Results are presented as arbitrary units normalized for β-tubulin. p<0.05: * <i>vs</i> limb.</p

Wnt ligands expression pattern in early stages of chick lung development.

<p>Representative examples of <i>in situ</i> hybridization of <i>wnt-1</i> (A–D), <i>wnt-2b</i> (E–H), <i>wnt-3a</i> (I–L), <i>wnt-5a</i> (M–P), <i>wnt-7b</i> (Q–T), <i>wnt-9a</i> (U–X) of stage b1, b2 and b3; n = 15 per stage. Open arrowhead – medial mesenchyme. Black arrow – proximal mesenchyme. Dagger – proximal epithelium. Asterisk – epithelial tip of the main bronchus. Dashed black arrow – secondary buds epithelium. Magnification: whole mount, 5×; slide sections, 20×. The black rectangle in images B, E, K, O, R and W indicate the region shown in corresponding slide section.</p

<i>In vitro</i> Wnt signaling inhibition (PK115-584) and branching analysis of lung explants.

<p>Representative examples of stage b2 lung explant culture, at D0∶0h (<b>A, D, G,</b>) and D2∶48h (<b>B, E, H</b>) treated with DMSO (A, B), 1 µM (D, E) and 2.5 µM (G, H) and probed with <i>axin2</i> (<b>C, F, I</b>); n = 4. Magnification: A, B, D, E, G, H –4x; C, F, I –5x. <b>M</b>: Branching analysis of stage b2 (n≥14 for each condition) explants treated with DMSO and PK115-584 (1 and 2.5 µM). Results are expressed as D2/D0 ratio. Data is represented as mean ± SEM. p<0.001: * <i>vs</i> DMSO, § <i>vs</i> 1 µM of PK115-584.</p

Schematic diagram of Wnt ligands expression pattern in a b3 stage lung.

<p>For simplicity, image was divided in half in order to avoid color overlay. Orange: <i>wnt-1</i> and <i>wnt-3a</i> expression; turquoise: <i>wnt-2b</i> expression; yellow: <i>wnt-5</i>a expression; dark blue: <i>wnt-7b</i> expression; purple: <i>wnt-9a</i> expression. Dual colors highlight different levels of expression.</p

<i>In vitro</i> Wnt signaling inhibition (PK115-584) and <i>β-catenin</i> expression.

<p>Representative examples of stage b2 lung explant culture, at D0∶0h (A, D, G) and D2∶48h (B, E, H) treated with DMSO (A, B), 1 µM (D, E) and 2.5 µM (G, H) and probed with <i>β-catenin</i> (C, F, I); n = 4 for each stage. Magnification: A, B, D, E, G, H –4x; C, F, I –5x.</p

Wnt signaling members’ expression pattern in early stages of chick lung development.

<p>Representative examples of <i>in situ</i> hybridization of <i>lrp5</i> (A–D), <i>lrp6</i> (E–H), s<i>frp1</i> (I–L), <i>dkk1</i> (M–P), <i>β-catenin</i> (Q–T), <i>axin2</i> (U–X) of stage b1, b2 and b3; n = 15 per stage. Asterisk – epithelial tip of the main bronchus. Dark arrowhead – periepithelial mesenchyme of secondary buds. Double dagger – mesothelium. Dashed black arrow – secondary buds epithelium. Open arrowhead – medial mesenchyme. Magnification: whole mount, 5×; slide sections, 20×. The black rectangle in images B, F, J, N, S and V indicate the region shown in corresponding slide section.</p