Fig. 4. The gene expression pattern underlying the regulation of AV canal formation and the possible regulation of Tbx2 by both Hesr1 and Hesr2. (A-F) Expression of the AV myocardium-specific markers, Bmp2 (A-C) and Tbx2 (D-F), were examined in wild-type (A,D), Hesr1-ME (B,E) and Hesr2-ME (C,F) mouse embryos (E9.5) by in situ hybridization. The reduction or lack of an AV canal was evident in these misexpressing hearts (B,C,E,F). (G-N) X-Gal staining of transgenic embryos containing either a 2.5 kb (Tbx2-Xho-nlacZ) (G,H) or 6 kb (Tbx2-D3-nlacZ) (I-N) upstream region of Tbx2 in wild-type (G-J), Hesr1-ME (K,L) or Hesr2-ME (M,N) mouse hearts. The heart regions shown in G,I,K,M are magnified in H,J,L,N, respectively. The brackets in A,B,D indicate the AV canal and the arrowheads in C,E,F indicate the AV boundaries. LA, left atrium; LV, left ventricle. (O) Schematic of the luciferase or LacZ reporter constructs harboring the Tbx2 upstream regions. A region containing Smad-binding sites is present 2.9 kb upstream from the first ATG of the Tbx2 gene. (P) Reporter assay using the Tbx2-D3 luciferase construct (shown in P) in NIH3T3 cells. Luciferase activity was assessed with or without the empty vector (pCDNA), 6xMyc-Hesr1 or 6xMyc-Hesr2, in the presence or absence of the constitutive active (CA) or kinase-dead (KD) form of Alk3 and Smad5.