Description
The Wnt signaling pathway is an intercellular singling mechanism required for a plethora of biological processes throughout an organism’s lifespan. Despite decades of research, this pathway is still largely misunderstood. Apart from its role in normal growth and homeostasis, Wnt signaling is also associated with numerous types of cancer. This underscores the current need to better understand how this cellular pathway functions. We have previously identified a specific Wnt signal between Fzd9b receptor with its ligand Wnt9a, which is important for proliferation of hematopoietic stem cells and cannot be rescued by other Wnts or Fzd proteins. We are now focused on deciphering mechanisms involved in transducing the signal to the cytoplasm once the Wnt9a ligand binds the Fzd9b receptor. We previously determined that the third intracellular loop of Fzd9b (ICL3) is important for its activity with Wnt9a, and that ubiquitination machinery is recruited to this portion of the receptor upon binding with the Wnt9a ligand. Here, we mapped the requirements for lysine residues in Fzd9b ICL3. Additionally, in a screen of ubiquitin modifiers, we found that Ubiquitin Specific Peptidase 8 (USP8) activates the Wnt9a/Fzd9b pathway, and that this activation requires K437. Apart from needing to better understand Wnt signaling itself, more accurate model organisms that can be used for high-throughput genetic and drug screens are necessary to identify mechanisms that can be used to target Wnt signals supportive of cancer. Here, we tested the CRISPR/Cas9 components that will be used in the near future to generate a zebrafish model of acute myeloid leukemia and determined that double-stranded breaks are generated at the target genomic locus. Additionally, the activity of the oncoprotein that will be expressed in this zebrafish model has been validated in an in vitro model. This leukemia zebrafish model will eventually provide a platform in which to study how Wnt activity promotes this cancer type as well as for the generation of targeted cancer therapies.
