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First published online 30 March 2005
doi: 10.1242/dev.01795

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Fgf10 expression identifies parabronchial smooth muscle cell progenitors and is required for their entry into the smooth muscle cell lineage

Arnaud A. Mailleux^1,*, Robert Kelly², Jacqueline M. Veltmaat, Stijn P. De Langhe^3,, Stephane Zaffran², Jean Paul Thiery¹ and Saverio Bellusci^1,,

¹ UMR144-CNRS/Institut Curie, 75248 Paris cedex 05, France
² URA 2578 CNRS, Department of Developmental Biology, Institut Pasteur, 25 rue du Dr Roux, 75015 Paris, France
³ Department for Molecular Biomedical Research, Flanders Interuniversity Institute for Biotechnology (VIB)-Ghent University, Technologiepark 927, B-9052 Ghent, Zwijnaarde, Belgium

View larger version (66K): [in a new window]   Fig. 1. Mlc1v-nLacZ-24+/– expression recapitulates Fgf10 expression. (A) Fgf10 expression by whole-mount in-situ hybridization in E10.5 wild-type control lungs. Note the expression in the right distal lung mesenchyme (black arrows), the thyroid and the developing stomach. (B) X-gal-stained E10.5 Mlc1v-nLacZ-24+/– lungs recapitulate the Fgf10 expression pattern at this stage in the distal lung mesenchyme (black arrow) and the thyroid. (C) Fgf10 expression by whole-mount in-situ hybridization in E11.5 wild-type control lungs. Note the expression in the left distal lung mesenchyme (white dotted box). (D) X-gal staining of E11.5 Mlc1v-nLacZ-24+/– lungs recapitulates the Fgf10 expression pattern at this stage, except in the left lobe (white dotted box). (E) Fgf10 expression by whole-mount in-situ hybridization in E12.5 wild-type control lungs. Note the expression in the distal lung mesenchyme (black arrows). The area in the dotted box is magnified in (F). (G) LacZ expression at RNA level in Mlc1v-nLacZ-24+/– lungs. (H) LacZ is expressed at the tip of the accessory lobe recapitulating the Fgf10 pattern (dotted box in G). Notice the absence of Fgf10 expression close to the epithelium (small double white arrow) (I) ß-galactosidase activity shown by X-gal staining is found in the distal mesenchyme (white arrow) and at the level of the bronchi of Mlc1v-nLacZ-24+/– lungs (small white arrows). Note that X-gal staining is now present in the mesenchyme of the left lobe. Note also that ß-galactosidase-positive cells are not detected in the primary bronchi (black arrows). (J) High magnification of the accessory lobe (dotted box in I). (K) Fgf10 expression at RNA level by whole-mount in-situ hybridization in E14.5 wild-type control lungs. Note the expression in the mesenchyme at the periphery of the lobes. (L) X-gal staining of E14.5 Mlc1v-nLacZ-24+/– lungs showing LacZ expression at the periphery of the lobes similar to the Fgf10 expression pattern. Note ß-gal expression at the level of the bronchi (black arrow). (M) High magnification of the surface of the cranial lobe shown in L. (N-Q) Control E11.5 Mlc1v-nLacZ-24+/– lung grown in absence of cyclopamine. (O) After 28 hours in culture new branches are formed. Note that the distal epithelium is not dilated (arrow). (P) X-gal staining of the cultured lung shown in O. ß-gal expression is found in the distal mesenchyme. (Q) Vibratome section through the left lobe shown by the arrow in P. Note the low level of ß-gal expression. (R-U) E11.5 Mlc1v-nLacZ-24+/– lung grown in presence of 5 µmol/l of cyclopamine. (S) After 28 hours of culture the lung exhibits dilated end buds (arrow). (T) X-gal staining of the cultured lung shown in S. Note the increase in ß-gal expression throughout the lung in comparison with the lung grown in the absence of cyclopamine shown in P. (U) Vibratome section through the left lobe of the lung shown by the arrow in T. Note the marked increase in LacZ expression compared with the untreated lung (Q). Scale bar: 110 µm in A,B; 180 µm in C,D; 210 µm in E,G,I; 105 µm in F,H,J; 435 µm in K,L; 80 µm in M; 175 µm in J; 250 µm in N,O; 300 µm in O,S; 190 µm in P,T; 50 µm in Q,U. acc, accessory lobe; br, bronchus; cont, control; cran, cranial lobe; st, stomach; th, thyroid; tr, trachea.

View larger version (122K): [in a new window]   Fig. 2. Fgf10-positive mesenchymal cells give rise to parabronchial smooth muscle cells. (A-D) Timecourse of ß-galactosidase expression in the accessory lobe between E11.5 and 12.5, showing the progressive extension of LacZ expression from distal mesenchyme (asterisk) toward the epithelium (arrow). Note the patchy expression of ß-galactosidase around the secondary bronchi at E12.5 (D). (E) A saggital vibratome section through an E12.5 Fgf10LacZ/+ accessory lobe showing almost continuous flow of ß-galactosidase expression in the distal mesenchyme (asterisk) and around the distal and proximal epithelium (arrows). Note that not all the cells around the epithelium of the secondary bronchi are positive for ß-galactosidase (red arrows). (E') A transversal section in secondary bronchus located in the most proximal part of E13.5LacZ/+ accessory lobe show that ß-galactosidase expression is found in the mesenchyme all around the epithelium. (F) Saggital section of E13.5 Fgf10LacZ/+ accessory lobe showing a continuous layer of ß-galactosidase-positive cells around the secondary bronchus. (G) Co-localization by immunofluorescence of ß-galactosidase (in green; note the nuclear signal) and -SMA (in red). (H) Whole-mount immunohistochemistry with anti-cytokeratin (in red) and anti-SMA (in green) antibodies on E12.5 lung. Note the presence of -SMA expression around the bronchi (small white arrows) and the absence of -SMA expression in the ventral part of the trachea (white arrow). (I) High magnification of the accessory lobe shown in H, demonstrating the absence of -SMA expression in the most distal part of the lung. Scale bars: 210 µm in A,B; 240 µm in C; 270 µm in D; 120 µm in E,F; 450 µm in H; 200 µm in I.

View larger version (131K): [in a new window]   Fig. 3. Fgf10-positive mesenchymal cells relocate around the bronchial epithelium. (A-D) ß-gal expression in the accessory lobe of unmanipulated Fgf10LacZ/+ lung after 48 hours of culture. (D) Vibratome section of the accessory lobe shown in C (dotted box). Notice the presence of ß-galactosidase-positive cells around the proximal epithelium (double arrow). (E-H) Grafting of Fgf10LacZ/+ mesenchyme (in blue) on wild-type lungs. (H) Vibratome section of the accessory lobe shown in G (dotted box). Note the presence of ß-galactosidase-positive cells around the proximal epithelium. The gray dotted line underlines the epithelium. (I-L) Ablation of the distal Fgf10LacZ/+ accessory lobe mesenchyme and grafting of equivalent wild-type mesenchyme (green) results in a drastic decrease of ß-gal expression around the bronchi (K,L). (L) Vibratome section of the accessory lobe shown in K (dotted box). Notice the absence of ß-gal-positive cells around epithelium. The epithelium is outlined by the dotted line. (M-N) Time-lapse sequence of E12 accessory lobes after the auto-grafting of CMFDA labeled distal mesenchyme (which includes the mesothelium). (M) Twenty-five-hour time-lapse fluorescence showing that mesenchymal cells are not actively migrating along the epithelium. (N) Phase-contrast time-lapse sequence of M, showing the outgrowth and budding of the epithelium. acc, accessory lobe; Bright f, Bright Field. Scale bar: 190 µm in A,B,E,F,I,J; 165 µm in C,G,K; 45 µm in D,H,L; 60 µm in M,N.

View larger version (97K): [in a new window]   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 Fgf10LacZ/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 Fgf10LacZ/– compared with Fgf10LacZ/+ 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 Fgf10LacZ/+ is set at 100% and used as a reference. Note the graded decrease in the expression of Fgf10 in Fgf10+/– (22%) and Fgf10LacZ/– (64%). (D,E) Decreased lung branching in E12.5 Fgf10LacZ/– 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 Fgf10LacZ/– (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 Fgf10LacZ/– lung. (H-K) Immunohistochemistry with -SMA antibody on E17.5 wild-type control (H) and Fgf10LacZ/– 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 Fgf10LacZ/+ 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 Fgf10LacZ/– 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.

View larger version (119K): [in a new window]   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 Fgf10LacZ/– 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 Fgf10LacZ/– 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 Fgf10LacZ/– compared with Fgf10LacZ/+ 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 Fgf10LacZ/+ lung are set to 100% and used as a reference. Note the decreased expression of Sprouty2 and Bmp4 in Fgf10LacZ/– 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 Fgf10LacZ/+ 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 Flk1LacZ/+ 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 Flk1LacZ/+ 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.