Figure 5. Notch modulates Wnt pathway transcriptional activity in both Drosophila and Vertebrate cells.

(A)Diagram of the different Notch molecules used in this study. (B) Loss of Notch function in Drosophila clone 8 (cl8) cells results in ectopic activation of Wnt signalling. Cells were transfected with the TOP12 reporter and a Renilla luciferase standard in the presence of no dsRNA, GFP dsRNA or Notch dsRNA, as shown. Ectopic activation of the Wnt signalling pathway was observed in cl8 cells in the presence of Notch dsRNA (103.6 fold activation compared to treatment with no dsRNA) but not GFP dsRNA (1.1 fold activation). (C , D) Gain of function of Notch in Drosophila cells results in repression of ectopically activated Wnt signalling. SL2 cells (C), or S2R⁺ cells (D) were transfected with the TOP12 reporter and Renilla standard, Wnt signalling was induced with an oncogenic form of ß-catenin, S37A β-catenin (Schweizer and Varmus, 2003). In the presence of a membrane tethered form of Notch (TNotch) the level of ectopic Wnt signalling was significantly reduced (C, D). (E-H) Gain of function of mouse Notch 1 results in repression of ectopic Wnt signalling induced by Wnt 1, Dsh and β-catenin in HEK-293T cells. Cells were transfected with the TOPFLASH reporter and a Renilla standard. Ectopic Wnt signalling was induced with Wnt1, delN-LRP6), Dishevelled, β-catenin or LEF1-VP16. In these assays two forms of Notch 1 were utilised; delN-N1 and LNR-N1(see A). DelN-N1 lacks the EGF and LN repeats and cleaves spontaneously to release the NICD domain of Notch1 as shown by the strong activation of the CBF1 reporter in HEK-293T cells (H), activation of the CBF1 reporter is approximately equivalent to that generated by an activated form of CBF1, CBF1-VP16. LNR-N1 is a membrane tethered form of N1 which lacks the EGF repeats but retains the LN repeats, that rarely cleaves as shown by the weak activation of the CBF1 reporter in HEK-293T cells (H and see also (Mumm et al., 2000)). In these experiments, canonical inhibition of Wnt signalling was effected by expression of ExFz8, which inhibits Wnt signalling by titrating Wnt (Brennan et al., 2004) but has no direct effect on the intracellular activation of the pathway. Small effects of ExFz8 on the endogenous Wnt signalling are visible, such effects have previously been reported by Suzuki et al.(Suzuki et al., 2004) (G). Both forms of Notch are capable of significantly repressing ectopic Wnt signalling induced by Wnt1, Dsh, and β-catenin (E and F), LNR-N1 effects also extended to ectopic Wnt signalling induced by delN-LRP6 and LEF1-VP16. On the other hand, ExFz8 repressed ectopic Wnt signalling induced by Wnt1 but had no effect intracellular mediators of Wnt signalling (G).