<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

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

<p>(A) Western blot analysis of phospho-LRP6 (Ser1490) and total LRP6 in stage b1, b2 and b3 lungs, and stage 24 limb (as positive control). Control loading was performed using β-tubulin (55 kDa). Phospho-Ser1490 and total LRP6 correspond to 190 kDa. (B) Semi-quantitative analysis for phospho-Ser1490 and total LRP6. Results are presented as arbitrary units normalized for β-tubulin.</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

Gp130 receptor expression pattern during fetal lung development.

<p>Gp130 receptor is expressed throughout all stages of lung development studied, from early 13.5 dpc until late 21.5 dpc and also in the adult. Representative immunohistochemistry staining of (A–E) developing lung and (F) adult lung. Original magnification ×100.</p