Hmga2−/−mice embryonic lung showed enhanced canonical WNT signaling. (A) Enriched cellular pathways (P <0.05) after Affymetrix microarray-based transcriptome analysis of Hmga2−/− E18.5 lung when compared to Hmga2+/+. Left, Kyoto Encyclopedia of Genes and Genomes pathway analysis performed using DAVID bioinformatics tool. Green, reduced expression (fold change <1.2); magenta, increased expression (fold change ≥1.2); repli, replication; ma, maturation; JAK-STA, Jak-STAT signaling; Toll lik, Toll-like-receptor signaling; RIG, RIG-like-receptor signaling. Right, Heat map was done using DNAStar Arraystar 11.0 and represents expression of genes involved in the WNT signaling pathway in Hmga2+/+ and Hmag2−/− E18.5 lung. Green, low expression; magenta, high expression; FC, fold change. (B) Hmga2-KO enhanced beta-catenin/TCF/LEF reporter activity in E14.5 lungs of the BAT-GAL transgenic mice. Representative (n = 5) BAT-GAL staining (blue color) shows activated WNT signaling. Scale bars, 2 mm. (C) Hmga2-KO increased levels of activated-beta-catenin (ABC) and phosphorylated LRP6 (pLRP6). Protein extracts from WT or Hmga2-KO E18.5 lungs were analyzed by western blot using the indicated antibodies. Lanes 2 and 3 are biological duplicates. (D) Hmga2-KO enhanced expression of canonical WNT pathway markers. Expression analysis of the indicated genes as in Figure 2F. (E) Hmga2-KO increased protein levels of canonical WNT pathway markers. Protein extracts were analyzed as in C using the indicated antibodies. (F) Hmga2-KO enhanced expression of secreted WNT glycoprotein genes Wnt7b, Wnt2b and Wnt11. Expression analysis as in Figure 2F. (G) Hmga2-KO increased nuclear localization of CTNNB1 in cells of the epithelium (epi) and the mesenchyme (mes) of the embryonic lung. Sections of E14.5 lung of WT and Hmga2-KO mice were analyzed as in Figure 2C using KRT- and CTNNB1- specific antibodies. Square shows details at higher magnification on the right. Nuclear staining with DAPI (blue). Arrows indicate nuclear CTNNB1. Dashed line shows epithelium-mesenchyme-boundary. Scale bars, 40 and 10 μm (right).