Abstract 26: Tcf7l1 Intrinsically Contributes to Cardiac Lineage Establishment

Abstract

T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) proteins are critical downstream effectors of the canonical Wnt pathway. Tcf7l1 is the most abundant among the four TCF/LEF proteins and acts as a key regulator of self-renewal in ESCs. It negatively modulates the expression level of pluripotent genes, and prepares the epiblast for transition to lineage specification. Tcf7l1-/- embryos show a range of defects related to axis misinduction. Some severely affected embryos lack the heart and somites. Because of defective axis induction, whether Tcf7l1 intrinsically contributes to cardiac development is unknown. Earlier work in our lab established that TCF/LEF proteins cooperate with Oct4 in driving the transcription of the cardiac mesoderm factor Mesp1, but the responsible TCF/LEF protein is not identified. Here, we have built ESC systems to conditionally manipulate Tcf7l1 expression in a Tcf7l1-/- background. A combination of tTA/tet-O-Tcf7l1 transgenes allows us to temporally “knockout” Tcf7l1 with doxycycline, while a combination of rtTA/tet-O-Tcf7l1 transgenes allows us to temporally activate Tcf7l1 expression with doxycycline. We found that “knockout” of Tcf7l1 on day 4 during ESC differentiation caused decreased expression in cardiac genes including Tbx5, Nkx2-5, and α-MHC, without affecting the expression of Goosecoid, Eomes, and Brachyury, markers of mesendoderm development. Tcf7l1-/- ESCs showed slower progression toward mesoderm formation, but temporally activated Tcf7l1 induced cardiac gene expression after mesoderm was formed. Tcf7l1 ChIP-PCR demonstrated that Tcf7l1 bound to cis-regulatory regions of core cardiac transcription factor genes. Our data suggest that Tcf7l1 intrinsically contributes to cardiac lineage development through activating Mesp1 and downstream cardiac transcription factors.