There are many ways to study the effects of enhanced canonical Wnt signalling in vitro. Three of the most common methods used are; the administration of recombinant Wnt ligands or Wnt-conditioned media to the cell culture, over- expression or knockdown of pathway components, and stabilisation of β-catenin though GSK3β inhibition. However, there are pros and cons for each method. Whilst using recombinant Wnt ligands should in principle supply the most biologically relevant levels of canonical Wnt activation, there are multiple problems associated with their use. Firstly, the availability of bioactive recombinant Wnt ligands is limited. While this is continuously improving with the generation of new ligands, there is the added complexity of ligand-specific effects, an area which is still relatively poorly understood. As mentioned previously, Wnt ligands can signal through multiple pathways, the canonical pathway, of interest here, and the non- canonical pathways. This offers the first level of complexity as several ligands are thought to be active through both pathways, while others are not clear (Mikels & Nusse, 2006). A second level of complexity is due to the current poor understanding of the differing effects of known canonical Wnt ligands in different situations, and the frizzled receptor and LRP co-receptor combinations to which they bind. With 19 Wnt ligands, and 10 Frizzled receptors (Mikels & Nusse, 2006), in combination with one of two co-receptors, one cannot even be certain that the cell of interest in any particular situation will be able to respond to the chosen ligand. This instantly makes it very difficult to draw conclusions about the ability of a particular ligand to generate a canonical Wnt response, and therefore the response of the cells to the Wnt stimulus.
In an attempt to circumvent these problems of ligand availability and specificity, manipulation of the pathway by altering the expression of key signalling molecules has been an attractive alternative. This can be done through the over-expression of positive regulators of the pathway; such as Wnt ligands (Wright et al, 1999), and β-catenin (Gould et al, 2007; Kim et al, 2006), or the down regulation of negative regulators; such as GSK3β (Cho et al, 2009), or DKK1 (Fleming et al, 2008). While
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this does offer strong stimulation of the canonical Wnt pathway as a whole, it allows for little regulation of the degree or duration of the activation.
The final method described here is the use of small molecule GSK3 inhibitors, to prevent the phosphorylation of β-catenin and its subsequent degradation. This allows for a more controlled stimulation of the canonical Wnt pathways, and inhibitors can be added at a range of concentrations for different time periods. Furthermore they are much more translatable for therapeutic uses. LiCl (de Boer et al, 2004; Etheridge et al, 2004; Spencer et al, 2006) and 6-bromoindirubin-3′-oxime (BIO) (Krause et al, 2010; Wang et al, 2009) are two commonly used inhibitors of GSK3 and are regularly used to mimic canonical Wnt signalling in vitro. However, they are not ideal, with off target effects and toxicity (Davies et al, 2000; Liu et al, 2011; Meijer et al, 2003). In addition to the non-specific effects of the inhibitors on GSK3β, GSK3β itself is not specific to the canonical Wnt signalling pathway, with involvement in hedgehog signalling (Jia et al, 2002) and regulation of the NFAT transcription factor (Kaytor & Orr, 2002).
There are multiple ways to study Wnt activation in vitro, most commonly the identification of nuclear β-catenin within the cell (Gambardella et al, 2011; Krause et al, 2010; Spencer et al, 2006), or increased stable dephosphorylated β-catenin levels (Boland et al, 2004; Krause et al, 2010; Kulkarni et al, 2006). More specific analysis of canonical Wnt activation examines the degree of TCF/LEF1 transcription factor activity (Boland et al, 2004; Etheridge et al, 2004; Kulkarni et al, 2006). The family of TCF/LEF1 transcription factors is the ultimate response to increased canonical Wnt signalling, generating the Wnt-dependent changes in gene expression. Therefore, the degree of activity of this transcription factor family gives an indication of the functional canonical Wnt response generation by whichever stimulation.
As mentioned previously, canonical Wnt stimulation is an appealing therapeutic target, and any method of stimulation must therefore also be able to generate a functional canonical Wnt response in vivo. One body of research which has provided a valuable method for studying the ability of compounds to stimulate canonical Wnt signalling in vivo is the Xenopus model of embryonic development.
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Spemann et al first identified the importance of the Spemann organiser in developmental organisation in 1924 and showed that grafting a second organiser region onto the ventral side of the embryo caused the duplication of the embryonal axis (Spemann H., 1924). The signalling mechanisms that underlie the initial positioning of the organiser region are now known (Figure 3.1.1) and together, they form the principles of the in vivo canonical Wnt activation assay (Kuhl & Pandur, 2008). Briefly, during the blastula stage of embryonic development, there is an accumulation of β-catenin in the dorsal side of the embryo that occurs in response to canonical Wnt signalling. This, in combination with VegT and Vg1 signals from the vegetal region, leads to the positioning of the Nieuwkoop centre. The Nieuwkoop centre releases Xenopus Nodal related factors which induce the formation of the Spemann organiser (De Robertis & Kuroda, 2004; Takahashi et al, 2000).
The canonical Wnt activation assay is carried out by the injection of the proposed canonical Wnt stimulus into the ventral side of the embryo. If successful in stimulating the canonical Wnt pathway, this leads to the generation a second area of increased β-catenin. Which, in turn, leads to the duplication of the various organiser regions, and results in the duplication of the embryonal axis (Figure 3.1.1), providing a clear readout for canonical Wnt stimulation. The accumulation of nuclear β- catenin also causes the formation of the blastula Chordin and Noggin expression (BCNE) centre, in the dorsal animal cap and marginal zone above the Nieuwkoop centre, resulting in the expression of BMP inhibitors, such as chordin and noggin (Wessely et al, 2001), which can be used as markers for induced β-catenin.
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Figure 3.1.1. Schematic of Xenopus embryonic patterning and duplication by Canonical Wnt
Injection of canonical Wnt agonists in the ventral side of the embryos leads to the duplication of the β-catenin accumulation, leading to the formation of second Nieuwkoop centre, and therefore Spemann organiser, which ultimately leads to the duplication of the embryonal axis.
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