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Fig. 4. Reduction of Fgf10 expression in the lung leads to a decrease in SMA expression around the bronchi. (A-B) Fgf10 expression shown by whole-mount in-situ hybridization in E10.5 wild-type (A) and Fgf10^LacZ/LacZ (B) embryos. Note the reduction in Fgf10 expression in the limbs (black arrows) and in the lung (white arrow). (C) Upper part: semi-quantitative amplification course by PCR on gel electrophoresis, respectively 1 (cycle 25), 8 (cycle 28) and 128 (cycle 32) amplification rate, showing decreased Fgf10 mRNA level in Fgf10^LacZ/– compared with Fgf10^LacZ/+ or Fgf10^+/– lungs at E14.5. Lower part: quantification of Fgf10 expression by densitometry at 128 amplification rate. Ratio of Fgf10 expression to tubulin in Fgf10^LacZ/+ is set at 100% and used as a reference. Note the graded decrease in the expression of Fgf10 in Fgf10^+/– (22%) and Fgf10^LacZ/– (64%). (D,E) Decreased lung branching in E12.5 Fgf10^LacZ/– lungs. High magnification on the left lobe shows that E12.5 mutant lung (E) is less branched (four buds) than control lobe (eight buds) (D). The epithelium is outlined by the gray dotted line. (F-G) Whole-mount immunohistochemistry with -SMA (in green) and cytokeratin (in red) antibodies in control (F) and Fgf10^LacZ/– (G) lungs at E12.5. The intensity of pixels representing the expression of -SMA around the right and left bronchi is quantified. A significant reduction (approximately 40% for the right secondary bronchi and 90% for the left secondary bronchi) is observed in the Fgf10^LacZ/– lung. (H-K) Immunohistochemistry with -SMA antibody on E17.5 wild-type control (H) and Fgf10^LacZ/– lungs (I). (J) Higher magnification of the dotted box shown in H. Note the labeled smooth muscle cells around the bronchi. (K) Higher magnification of the dotted box shown in (I). Note the drastic decrease in -SMA (white arrow) expression around the bronchi. (L) X-gal staining of E13.5 Fgf10^LacZ/+ lung, showing significant ß-galactosidase signal around the epithelium of the secondary bronchi of the accessory lobe. (M) X-gal staining under the same conditions of E13.5 Fgf10^LacZ/– lung, showing strong decrease in ß-galactosidase-expressing cells around the bronchial epithelium of the secondary bronchi of the accessory lobe as well as accumulation of ß-galactosidase-expressing cells at the distal tip. Scale bar: 590 µm in A,B; 150 µm in D,E right part; 300 µm in D,E left part; 150 µm F,G; 60 µm in H,I; 15 µm in J,K; 100 µm in L,M. br, bronchi; smc, smooth muscle cells.

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