Fig. 8. Tbx20 directly binds and represses Isl1 in myocardium. (A-D) Isl1 is expressed throughout myocardium in Tbx20 mutants. (E) ChIP analysis revealed Tbx20 recruitment to region of Isl1 promoter with conserved T-box site (lane 1, primer P-842, P-581). ChIP analysis with primers against an unrelated promoter region revealed no Tbx20 recruitment (lane 2) (see Materials an methods for primers). No recruitment was found with beads, IgG or H₂O. (F) Co-transfection of Tbx20 expression vector (500 ng) with Isl1 promoter-luciferase reporter (100 ng) demonstrated repression of Isl1 by Tbx20. Repression was abrogated by mutation of the consensus T-box element within the Isl1 promoter. ^*P<0.05, paired t-test. (G-J) The nebulette promoter, expressed exclusively in myocardium during early embryogenesis (J.C., unpublished) was used to drive expression of Isl1 in transient transgenic mice. Whole-mount in situ hybridization (G,H) revealed expression of Isl1 throughout myocardium in transgenic mice (H). Hearts of transgenics appeared normal at E10.5. The left and right images in A-D,G,H show right and left side views. Corresponding sections are shown progressively from anterior to posterior (C,D; I,J). Arrows indicate the corresponding heart regions in wild type and mutant embryos.