<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 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

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

<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

TUNEL assay in chick lung explants.

<p>Representative examples of negative (A) and positive control (B), DMSO (C), 20 µM (D), 30 µM (E) and 40 µM (F) FH535 treated stage b2 explants; n = 4 per condition. Negative control explants were incubated without TUNEL mix solution. Positive control explants were incubated with DNase at 37°C for 90 minutes.</p

<i>In vitro</i> Wnt signaling inhibition and <i>fgf10</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>fgf10</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.</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

<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