Tbx1 regulates VEGFR3 expression via a conserved TBE in the endogenous VEGFR3 gene. (A) Schematic of the In^11-12TBE luciferase reporter. A mutant version of the reporter construct was generated by site-directed mutagenesis of the TBE located within the putative VEGFR3 enhancer (In^11-12TBE). (B) Cotransfection of JEG3 cells with the luciferase reporter and increasing amounts of Tbx1 expression vector increased activity of the luciferase reporter containing the wild-type enhancer (wt-TBE) but not the mutant enhancer (mut-TBE). Control cultures were transfected with empty expression vector. (C) PCR of chromatin immunoprecipitated from HUVECs. Antibodies used for immunoprecipitation are indicated above the panel. A chromatin sample immunoprecipitated with a nonspecific antibody (c-Myc) served as a negative control and the input sample as a positive control. Samples immunoprecipitated with anti-Tbx1 showed enrichment of the VEGFR3 enhancer containing the conserved TBE. (D–E′) Histological sections of the mesentery of transgenic embryos carrying the wild-type (D and E) or mutant TBE (D’ and E’) enhancer construct at E15.5. Colocalization of β-gal expression and anti-Vegfr3 immunostaining was seen in mesenteric LECs of transgenic embryos carrying the wild-type TBE (D and E, arrowheads) but not with the mutated TBE (D’ and E’). Arrows indicate the mesenteric artery (A). V, mesenteric vein; L, lymphatic vessel. Bars, 100 µm.