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First published online 18 July 2007
doi: 10.1242/dev.006221

Development 134, 2969-2979 (2007)
Published by The Company of Biologists 2007
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Inhibition of Tgfß signaling by endogenous retinoic acid is essential for primary lung bud induction

Felicia Chen1, Tushar J. Desai2, Jun Qian1, Karen Niederreither3, Jining Lü1 and Wellington V. Cardoso1,*

1 Pulmonary Center, Boston University School of Medicine, Boston, MA 02118, USA.
2 Biochemistry Department, Stanford University, Stanford, CA 94305, USA.
3 Baylor College of Medicine, Houston, TX 77030, USA.


Figure 1
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  		Fig. 1. The Tgfß pathway is regulated by RA at the onset of lung development. (A) Western blotting of cultured mouse foregut explants (E8.5 plus 24 hours) revealing increased phosphorylation of Smad2 (pSmad2) in RA-deficient conditions (lane 3, BMS493-treated WT; lane 4, non-RA-supplemented Raldh2-/-) as compared with RA-sufficient conditions (lane 2, WT control; lane 5, RA-supplemented Raldh2-/-). TGFß1-treated WT foregut is used as a positive control (lane 1). Total Smad2 (tSmad2) is used for normalization. (B) Real-time PCR showing rapid dose-dependent induction of Tgfbi in TGFß1-treated lung mesenchymal cells (MLg cells; asterisks indicate P<0.05 by Student's t-test). (C,G) Hematoxylin and Eosin (H&E) staining of paraffin-embedded sections showing lung bud formation in the control, but not in the BMS493-treated foregut culture. (D,H) RARElacZ expression is strong in control foregut cultures, but is dramatically suppressed by BMS493 treatment (asterisk marks the presumptive lung region in G,H). (E,F,I,J) Whole-mount in situ hybridization (WMISH) and immunostaining of control foreguts revealing a low level of Tgfbi mRNA (E, arrowheads) and Tgfbi protein (F, red) expression in the foregut mesoderm of the lung primordium. BMS493 treatment results in increased Tgfbi mRNA (I, arrowhead) and protein (J, red) in the mesoderm of the presumptive lung and stomach fields, and stronger pSmad2 signals, as compared with the control (F,J, green). Ht, heart; Lu, lung; St, stomach; Ctr, control. Scale bar: 300 µm in E.

 

Figure 2
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  		Fig. 2. Expression of Tgfbi, a target of the Tgfß pathway, under RA-sufficient and -deficient conditions. (A-C) X-Gal staining of E9.5 RARElacZ mouse embryo showing strong signals in the foregut region (A, between the dashed lines), where Tgfbi expression is minimal by WMISH (asterisk in B,C, between dashed lines). By contrast, Tgfbi signals are significantly stronger in the same region of the Raldh2-/- foregut in vivo (C, arrowhead), compared with a WT littermate. (D) WMISH showing high levels of expression of Tgfbi in a non-RA-supplemented Raldh2-/- foregut explant. The Tgfbi expression domain depicted in the boxed area includes the thyroid (Th) and the region where the lung failed to form. (E) WMISH of Nkx2.1 in Raldh2-/- control cultures. Asterisk (in the enhanced-contrast image of the explant, D, right; E) marks the presumptive lung field. (F,G) Tgfbi expression is dramatically reduced in Raldh2-/- foregut in which lung (Lu) bud formation was rescued by RA supplementation (F, arrowhead). H&E staining of paraffin-embedded section of RA-supplemented Raldh2-/- reveals bud formation in the presumptive lung region of the foregut (G, arrowhead). The presence of lung bud formation is further confirmed by WMISH of Nkx2.1 in RA-supplemented Raldh2-/- foregut (G, inset, arrowhead). Dotted lines outline the lung. (H) Real-time PCR showing downregulation of Tgfbi in RA-treated lung mesenchymal (MLg) cells (*, P<0.05 by Student's t-test). Ht, heart; St, stomach; Ctr, control. Scale bar: 300 µm in E.

 

Figure 3
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  		Fig. 3. Ctgf and Col1a2 are Tgfß targets upregulated in RA-deficient mouse foreguts. Each panel depicts WMISH (left) and the corresponding black and white enhanced-contrast image of the explants (right). Boxes outline the region that includes the lung domain. (A-D) WMISH reveals no Ctgf signals in the WT control foregut (A), but expression is significantly upregulated in the mesoderm at the presumptive lung region of the BMS493-treated foregut (B, asterisks) and non-RA-supplemented Raldh2-/- foregut (C, asterisks). Ctgf expression is markedly suppressed by RA supplementation in Raldh2-/- foregut (D). (E-H) Col1a2 expression is detected by WMISH in the mesoderm of WT control lung bud (E) and is also dramatically increased in BMS493-treated foreguts (F, asterisks) and in non-RA-supplemented Raldh2-/- foreguts (G, asterisks). Expression of Col1a2 is markedly downregulated by addition of exogenous RA in Raldh2-/- foregut (H). Ht, heart; Lu, lung; St, stomach; Ctr, control. Scale bar: 300 µm in C.

 

Figure 4
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  		Fig. 4. Hyperactivation of Tgfß signaling disrupts lung formation in the developing foregut. (A,B) Treatment of mouse foregut explants with recombinant TGFß1 results in lung agenesis (B, asterisk). (C,D) WMISH and immunostaining showing generalized induction of Tgfbi message (C) and protein (D), in TGFß1-treated foreguts. (E-H) PCNA staining showing abundant endodermal and mesodermal labeling in both control and TGFß1-treated foreguts (boxed regions in E,G are magnified in F,H). (I-L) WMISH showing Nkx2.1 and Sftpc in lung buds of controls (I,K, arrowheads), but not in the presumptive lung region of TGFß1-treated foreguts (J,L, asterisks). Ht, heart; Lu, lung; St, stomach; Th, thyroid; Ctr, control. Scale bar: 200 µm in E.

 

Figure 5
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  		Fig. 5. TGFß1 disrupts Fgf10 expression in the mouse foregut mesoderm. (A,B) X-Gal staining of RARElacZ foregut cultured in TGFß1-containing medium reveals strong RARElacZ signal at 3 hours (A) and 72 hours (B) in culture. (C,D) Fgf10 is expressed in control foreguts (C, arrowheads) and is suppressed in TGFß1-treated foreguts (D, asterisk). (E) Real-time PCR results showing the downregulation of Fgf10 in MLg cells by TGFß1 treatment (*, P<0.05 by Student's t-test). (F,G) FGF10-but not PBS-soaked heparin bead rescues Nkx2.1 expression and bud outgrowth in TGFß1-treated foreguts (G, arrowhead). Ht, heart; Lu, lung; Th, thyroid; Ctr, control. Scale bar: 250 µm in F.

 

Figure 6
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  		Fig. 6. Expression of Tgfß ligands and receptors in the mouse foregut and early lung. (A-H) WMISH of Tgfbr1 and Tgfbr2 showing that both receptors are diffusely expressed in the E8.5 foregut (A,E) and in control cultured foregut at 24 hours (B,F). By 72 hours in culture, strong Tgfbr1 (C) and Tgfbr2 (G) expression is detected in the endoderm and mesoderm of the lung region (boxed areas in C and G, magnified in L). At E12.5, both receptors are highly expressed in the distal lung in vivo (arrows). (I,J) WMISH of Fgf10 (I) and X-Gal staining of Fgf10lacZ (J) expression in E9.5 embryos reveal Fgf10 expression in the lung region (circled). (K) X-Gal staining of an Fgf10lacZ foregut explant at 72 hours showing strong lacZ expression in the mesenchyme associated with the distal lung buds. (L) Boxed areas from C, G and K, showing overlap of Fgf10lacZ, Tgfbr1 and Tgfbr2 expression in the mesenchyme at the lung field. (M) Histological section of WMISH specimens from L showing localization (arrowheads) of Tgfbr1 and Fgf10lacZ expression. (N-V) WMISH of Tgfb1-3. Freshly isolated E8.5 explants (N,Q,T) and control 24-hour cultures (O,R,U) show diffuse expression of these ligands in the foregut region, mostly in the mesoderm; signals are in some cases associated with cardiac and vascular structures (Tgfb1, arrowheads in N,O; Tgfb2, arrowhead in R) or endodermal structures (Tgfb2 in S), as previously reported. In the E12.5 lung, Tgfb1 is expressed in the subepithelial mesenchyme (P, arrowhead), Tgfb2 is mostly restricted to the distal epithelium (S, arrowhead), and Tgfb3 is present in mesothelial cells of the pleura and distal epithelium and mesenchyme (V, arrowhead). en, endoderm; me, mesoderm; Fg, foregut; Lu, lung; Ht, heart; St, stomach.

 

Figure 7
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  		Fig. 7. Effect of TGFß-blocking antibody on Tgfbi, Col1a2 and Ctgf expression and lung bud formation. Expression of Tgfbi (B) Col1a2 (D) is noticeably decreased in WT mouse foreguts treated with a pan-specific TGFß-blocking antibody (TAb, asterisks) when compared with foreguts cultured in control medium (A,C, arrowheads). Culturing Raldh2-/- foregut in TAb (F, asterisk) prevents the high-level Ctgf expression typically seen in the untreated Raldh2-/- foregut (E, arrowheads). Lung bud formation in WT foreguts is not affected by the treatment with TAb (B,D). Panels to the right are the corresponding enhanced-contrast images of the explants in B and D. Lu, lung; Ctr, control. Scale bar: 300 µm in B.

 

Figure 8
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  		Fig. 8. Blocking Tgfß signaling rescues bud formation and gene expression in the lung field of RA-deficient foregut. (A-D) BMS493-treated mouse foregut fails to induce lung bud formation (A, asterisks; E, asterisk, boxed region). However, treatment with a combination of BMS493 and pan-specific TGFß-blocking antibody (TAb) allows bud formation and strong Nkx2.1 signals are detected in the prospective lung region of the WT foregut (B, arrowheads). (C,D) Similarly, untreated Raldh2-/- foregut does not form lung buds under the control condition (C, asterisk), but budding and Nkx2.1 expression are partially rescued by TAb treatment (D, arrowheads). (E,F) In BMS493-treated WT foregut, Fgf10 mRNA is seen in the thyroid and pancreatic fields, but not in the prospective lung region (E, boxed area). In WT foregut treated with both BMS493 and TAb, there is strong Fgf10 expression (F, boxed area) associated with the rescued lung bud (F, arrowhead). Panels to the right are the corresponding enhanced-contrast images of the explants in E and F. Th, thyroid; Ht, heart; Lu, lung; Pa, pancreas; CAb, unrelated isotype-matched control antibody. Scale bar: 270 µm in A.

 

Figure 9
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  		Fig. 9. RA-Tgfß-Fgf10 interactions during primary lung bud formation in the mouse. (A) In an RA-sufficient foregut, endogenous RA maintains low levels of Tgfß signaling in the mesoderm of the lung field to allow Fgf10 expression and lung bud initiation. (B) Under conditions of RA-deficiency, Tgfß signaling is abnormally hyperactivated, thereby blocking Fgf10 expression and lung bud formation.

 




© The Company of Biologists Ltd 2007