Fig. 1. (A) Conditional control of Foxa2 gene targeting. In triple transgenic mice (SP-C-rtTA^-/tg, (tetO)₇CMV-Cre^-/tg, Foxa2^loxP/loxP and CCSP-rtTA^-/tg, (tetO)₇CMV-Cre^-/tg, Foxa2^loxP/loxP), rtTA is expressed in epithelial cells under the control of either the human SP-C or CCSP promoters. In the presence of doxycycline, rtTA binds to the (tetO)₇CMV promoter and activates the expression of Cre-recombinase, causing recombination and deletion of exon 3 in Foxa2^loxP/loxP mice, and producing Foxa2^/ mice. (B) Immunohistochemistry demonstrates Foxa2 deletion. Lung sections were prepared on PN16 for immunohistochemistry using anti-FOXA2 antibody. Triple transgenic mice, SP-C-rtTA^-/tg, (tetO)₇CMV-Cre^-/tg, Foxa2^loxP/loxP (B part B) and CCSP-rtTA^-/tg, (tetO)₇CMV-Cre^-/tg, Foxa2^loxP/loxP (B part D), and littermate control mice (B part A, B part C) were maintained on doxycycline from E0. Inserts are higher magnifications (x40) of the regions indicated by arrowheads. Nuclear FOXA2 staining was observed in epithelial cells of conducting and peripheral airways and alveoli, and was absent or decreased in Foxa2^/ mice. Goblet cell hyperplasia was observed in both SPC-rtTA and CCSP-rtTA Foxa2^/ mice (insets). Figures are representative of at least four individual mice. Scale bar, 50 µm. (C) RNA protection assay for estimation of Foxa2 mRNA. RNA protection assays were used to quantitate Foxa2 mRNA in lungs from SP-C-rtTA, Foxa2^/ and control littermates at E18.5 and compared with L32 mRNA. Dams were treated with doxycycline from E0 to E18.5.