How to activate and detect Wnt signaling
Researchers who want to detect active Wnt-ßcatenin signaling in cells have various tools available.
To measure Wnt signaling, one can use a variety of Wnt reporters. The TOP-flash assay (Molenaar 1996) is widely used and variants of TOP-Flash (SuperTop) are available from various sources including lentivirus-based reporters from the Nusse and Moon labs, through Addgene. One can transduce these reporters into cells, together with an appropriate control and measure the levels of Wnt-ßcatenin signaling by luciferase activity.
To measure Wnt signaling in intact animals, there are various reporter mouse lines available. There is a separate page on Wnt signaling tools in intact animals,
Most cells respond to Wnt signaling by an increase in the levels of beta-catenin. There are good antibodies to detect beta-catenin either on Western Blots or by staining (Transduction labs). There is also a monoclonal antibody to the non-phosphorylated ("activated") form of beta-catenin (Van Noort, 2002) available from Upstate. While it is known that beta-catenin is present in the nucleus of Wnt activated cells, detecting the protein by staining is not completely unequivocal and there are several situation where beta-catenin cannot be visualized in the nucleus, even though the protein is known to function there (Riggleman et al, 1990; Tolwinski and Wieschaus, 2004).
Another response to Wnt is an increase in the expression of Axin2, but as far as I know, there are no good antibodies available to Axin2.
To activate Wnt signaling, one can add Wnt protein, either in a purified form or as conditioned medium to cells. Cells producing active Wnt can be obtained from the ATCC (see also the reagents page). Comparing the Wnt conditioned medium to medium from control L cells is a reasonable way of controling for specificity but it should be realized that Wnt expression can lead to the induction of genes encoding other secreted factors (such as FGFs, see target genes) and that the conditioned medium may therefore contain such factors.
Whether cells respond to the particular Wnt will depend on the receptors expressed. There is something known about Wnt-Frizzled eceptor specificity. How cells will respond to Wnt signaling depends very much on the state of the cell; most of the Wnt target genes are cell type specific and only activated in certain tissues.
Several workers have used LiCl, an inhibitor of GSK3, to activate Wnt signaling (Klein and Melton, 1996). Keep in mind that GSK3 has other functions as well and that LiCl is therefore not very specific for just activating the Wnt pathway (Cohen and Goedert, 2004, Crabtree and Olson 2002).
Several other inhibitors of GSK3 have been described (Meijer 2004). Of these, the small molecule CHIR99021 is used widely to activate Wnt signaling in cells, including stem cells, embryonic stem cells (as part of the 2i condition, Silva 2008).
Blocking negative regulators of Wnt signaling, such as Axin and APC will also activate the pathway, and using RNAi is an obvious choice.
There are proteins unrelated to Wnt, such as norrin (Xu et al, 2004) and R-spondin (Kazanskaya 2004 ; Kim et al, 2005) that can also activate the Wnt pathway. Norrin binds to Frizzled4 (but only to that member of the Frizzled family); while R-spondin interacts with LGR receptors De Lau 2011, Carmon 2011 (Nam, 2006) and LRP6 (Wei, 2007). See the separate page on R-Spondin.
By gene transfer, including transfection, one can activate Wnt signaling, using either Wnt overexpression constructs or beta-catenin. The amino-terminal truncated form of beta-catenin works best in these assays (Reya et al, 2003)