The Wnt signalling pathway is a fundamental cell-to-cell communication system that plays a pivotal role in orchestrating diverse developmental processes across metazoans. There are thirteen subfamilies of Wnt ligands, seven of which are represented in the model organism Drosophila melanogaster. Similar expression of some Drosophila Wnt genes has been observed in several tissues, which suggests they may play overlapping roles, however, loss of certain Wnt genes such as Wnt6 appear to affect only certain tissues. This could suggest that Wnt ligands act in a combinatorial manner in Wnt signalling landscapes in different tissues, despite functional differences at the molecular level. Despite significant advances in our understanding of the Wnt pathway, the functional specificity of these diverse Wnt ligands remains enigmatic. This thesis aimed to address this knowledge gap, using the following different approaches. Sequences of the seven Drosophila Wnt ligands were compared to each other and protein structures for each of the seven Wnt ligands in Drosophila were predicted to identify any similarities and differences in the protein structures. Protein structures were modelled using templates from the existing crystallised protein of Xenopus Wnt8 and then compared with models created with artificial intelligence. Results from this chapter highlighted the existence of unique domains that vary in positioning across the seven Wnt genes. The seven Wnt ligands represented in Drosophila were then compared to predicted protein models across a range of animals that covers all major animal divisions including: Nematostella vectensis, Platynereis dumerilli, Mus Musculus , Aides aegypti , Tribolium castaneum Bicyclus anynana , and Parasteatoda tepidariorum . This work indicated that insertion predicted in Drosophila Wnts appears to be unique and mostly absent from the other orthologs. Tests were then undertaken to understand whether Drosophila wg can be functionally replaced by its ancient paralog Wnt6, thus functional evolution within the Wnt ligand family. Drosophila Wnt6 was found to be unable to functionally replace Drosophila wg. This indicates that specific protein domains present in Drosophila Wg, but not in Drosophila Wnt6, are essential for its functionality. To test if specific domains underlie Wg functionality, and thus validate or challenge the above statement, a series of chimeric Wg/Wnt6 proteins, which had domains swapped between these proteins, were tested for their ability to rescue Wg in Drosophila. It was observed that these chimerics were unable to rescue. Finally, the rescue capacity of the Wg orthologs from other species (Nematostella vectensis , Platynereis dumerilli, Mus Musculus , Aides aegypti , Tribolium castaneum Bicyclus anynana , and Parasteatoda tepidariorum ) were tested in Drosophila development including during wing development. This aimed to map the domains responsible for the functional differences between wg orthologs. However, none of the Wg orthologs could rescue indicated the capacity to rescue Drosophila Wg function. These results, taken together, suggest that protein domains present in Drosophila Wg may confer specific functionality to the ligand. In summary this thesis will aim to investigate the functional divergence of Wnt genes in Drosophila.
Permanent link to this resource: https://doi.org/10.24384/6bkm-tk19
Booth, Shamma Rattan
Supervisors: McGregor, Alistair
Department of Biological and Medical Sciences
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