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.