Fig. 5. Cripto is regulated by ß-catenin/Tcf in colon cancer cells and tumours. (A) Northern blot analysis of Cripto expression in Ls174T colon adenocarcinoma cells. Expression of Cripto in control Ls174T cells and in cells that express dominant-negative Tcf4 in a tetracycline-inducible manner is shown. Cripto expression was assessed before (-) and 12 hours after (+) induction with doxycyclin. RNA-loading control, 28s rRNA. (B) In situ hybridisation on consecutive sections of intestinal epithelium of Min (APC mutant) mice that contain an adenoma. The ß-catenin target gene conductin marks the crypts and the adenoma. Cripto and Tssc3 (see Table 1) are also upregulated in the adenoma. (C) Identity plot of murine and human genomic sequences in the region of the Cripto locus. The mouse sequence is indicated on the horizontal axis. The transcribed region of Cripto is indicated by a horizontal arrow, the position of Cripto exons by black boxes. The Cripto enhancer (8 kb upstream) and immediate 5' flanking regions are indicated by red and green boxes, respectively. Conserved sequence stretches between mouse and human sequences are indicated by the short horizontal lines (50% to 100% identity). Consensus sequences for Lef/Tcf binding sites (WWCAAAG) in the murine genome are indicated by vertical red lines. Sequences were aligned using the Pipmaker program (Schwartz et al., 2000). (D,E) Identification of ß-catenin/Tcf responsive elements in the Cripto enhancer by luciferase reporter gene assay, using a Tcf4-ß-catenin hybrid effector plasmid. ß-Catenin-responsive TOPflash and inactive FOPflash have been described previously (Molenaar et al., 1996). Mutations in the three Lef/Tcf-binding sites (3xMUT) are described in the Materials and methods.