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Wnt signaling pathway overview
- Wnt acts by attaching to the cell membrane receptor Frizzled, which then activates Dishevelled in the cytoplasm.
- Dishevelled blocks GSK-3 activity. GSK-3 is part of a complex which includes APC and Axin, which act to degrade -catenin.
- When GSK-3 is blocked, B-catenin is allowed to accumulate in the cytoplasm and then eventually it is translocated to the nucleus where it is involved in transcription.
- Axin is one of the important negative regulators of B-catenin that is described in one of the experiments below. Axin will act to degrade any B-catenin in the cytosol, preventing -catenin from translocating and thereby inhibiting transcription.
DEVELOPMENTAL EVENT THAT UTILIZES WNT PATHWAY
Wnt is involved in the limb bud initiation and development, and is studied specifically in vertebrate embryos in the paper Wnt Signals Control FGF- Dependent Limb Initiation and AER Induction in the Chick Embryo. In this paper, the Wnts studied are shown to act via the Wnt/β-catenin pathway explained above, in order to aid in limb initiation and development.
1ST experiment exploiting the Wnt pathway
1. The authors first look to see if the Wnts studied act through the canonical Wnt/β-catenin pathway, which leads to the activation of β-catenin. They conduct a western blot in order to visualize if β-catenin protein is present in the cytosol of chick embryos that have been injected with a retrovirus for the Wnts of interest. Results show that these Wnts do in fact utilize the canonical Wnt/β-catenin pathway by interacting to the cell membrane receptor, resulting in the activation of β-catenin, as the western blot shows cytosolic leveles of β-catenin present with these Wnts. The authors then inject embryos with a retrovirus encoding the activated form of β-catenin into the flank of embryos. The embryos are allowed to develop and then, through a skeletal staining, ectopic limbs were observed. These Wnts, utilizing the Wnt pathway to activate β-catenin, are therefore SUFFICIENT in ectopic limb formation.
2ND experiment exploiting the Wnt pathway
- 2. The authors want to also show if activated β-catenin is NECESSARY for limb initiation and development. As shown in the pathway, Axin acts as a negative regulator of β-catenin through degradation in the cytosol. Adenonoviruses encoding Axin were used in order to overexpress Axin in the flank of embryos. When expressed in the early stages of limb development (and skeletal staining as done above) it drastically interfered with limb development, in some cases not no limb skeletal elements were observed at all. Therefore β-catenin is shown to be necessary for limb initiation. Since the Wnts studied here have been shown to act through the Wnt/β-catenin pathway, the authors imply that Wnt is necessary for limb initiation.
- **Both #1 and 2 utilize viruses to overexpress β-catenin and Axin, while #1 initially uses Western blotting to look at expression of β-catenin protein