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Fig. 5. FGF10 regulates the establishment of the parabronchial smooth muscle cell lineage via upregulation of Bmp4 expression in the epithelium. (A) Treatment of lung SMC with either FGF1 or FGF7 or FGF10 (100 ng/ml) for 20 minutes. Only FGF1 induces the phosphorylation of 42/43 MAPK (upper bands). Total ERKs (after striping of the previous blot) and -SMA blots correspond to loading controls. (B) Sonic hedgehog expression in E14.5 normal and Fgf10^LacZ/– caudal lobes was used to outline the epithelial buds. Note that Shh expression is not significantly altered in the mutant lobe. (C) Bmp4 expression in wild-type and Fgf10^LacZ/– accessory lobes at E14.5, showing a lower expression level in the mutant lobe (white arrow). In the distal part, Bmp4 expression level is less decreased. (D) Sprouty2 expression in normal and mutant median lobes at E14.5, showing a lower expression level in the mutant lobe. (E) Semi-quantitative amplification course by PCR on gel electrophoresis. 1 (cycle 28), 8 (cycle 31) and 64 (cycle 34) amplification rate, respectively, showing decreased Spry2 and Bmp4 mRNA level in Fgf10^LacZ/– compared with Fgf10^LacZ/+ and Fgf10^+/– lungs at E14.5. Quantification of Spry2 and Bmp4 expression by densitometry at 64 and 8 amplification rate, respectively. Ratios of Spry2 to tubulin and Bmp4 to tubulin in Fgf10^LacZ/+ lung are set to 100% and used as a reference. Note the decreased expression of Sprouty2 and Bmp4 in Fgf10^LacZ/– lung compared to the reference (45% and 30% reduction, respectively). In both cases, Spry2 and Bmp4 expression levels were not decreased in Fgf10^+/– lungs (respectively 95% and 99% of Fgf10^LacZ/+ expression level). (F-I) Comparison of -SMA expression in wild-type, SpC-Shh and SpC-Bmp4 transgenic lungs at E16.5. (F) Control lung showing -SMA expression in the mesenchymal cells around the bronchial epithelium but excluded from the tip. (G) SpC-Shh lungs showing no disruption of -SMA expression. (H) High magnification of a wild-type lung showing -SMA expression around the bronchial epithelium and around the blood vessels. (I) -SMA expression in SpC-Bmp4 lungs showing a drastic increase of -SMA expression in the distal mesenchyme. Note the expanded epithelium, which is characteristic of the SpC-Bmp4 lungs. (J) Isolated E13.5 mesenchyme explants grown for 48 hours in Matrigel show no -SMA-expressing cells by immunohistochemistry. (K) Identical experiment with mesenchyme explants from Flk1^LacZ/+ lungs to show the presence of endothelial cells in the explant (arrow). (L) Addition of recombinant 100 ng/ml of recombinant BMP4 induces -SMA expression. (M) Identical experiment with Flk1^LacZ/+ mesenchymal explants shows the presence of endothelial cells within the BMP4-treated explant. Scale bar: 200 µm in B; 160 µm in C; 140 µm in D; 70 µm in F,G; 35 µm in H-K. br, bronchial epithelium; bv, blood vessels.

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