Evolution and functionality of Wnt genes in the fruit fly Drosophila melanogaster

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Wnt signalling regulates many biological processes during animal development, including cell fate specification, division and patterning. There are thirteen subfamilies of Wnt ligands in metazoans, seven of which are represented in the fruit fly Drosophila melanogaster. Most metazoan species have retained a large number of Wnts. Although some Wnt genes appear to have overlapping roles in Drosophila, it is thought they act cooperatively in Wnt landscapes and in some contexts they perform unique functions. To date, however, the molecular basis for the functional diversification of Wnt proteins and their individual interactions with receptors and other proteins remains unknown. For the project, I will use a novel reintegration system to identify the protein sequences responsible for functional divergence between Wnt genes. The CRISPR/Cas9 system will be applied for genomic engineering to delete specific genomic sequences and to simultaneously reintroduce exogenous DNA using targeting vectors. This system offers a remarkable opportunity to directly analyse and compare functions of Wnt ligands and hence test if the ligands can rescue each others functions to understand the molecular basis of their differences. Therefore, I will test if wg and Wnt6 can be replaced by each other in regards to their phenotypes after a gene loss in Drosophila. Next, I will analyse if Wnt10 and Wnt9 can rescue each others functions during embryogenesis. Since it is unlikely that one Wnt gene can completely and functionally replace another Wnt gene, I will reintroduce chimeric ligands of different Wnt genes to identify domains responsible for the diversification of two Wnt ligands. Thus, I will introduce chimeric wg-Wnt6 and Wnt9-Wnt10 genes, respectively, and assay for improved rescue. In the unlikely event that Wg and Wnt6 or Wnt9 and Wnt10 are fully functionally compatible, I will use the next closest related Wnt ligand Wnt8, and chimeric ligands thereof. This project will identify the molecular basis of functional differences among Wnt ligands, which will help to clarify how Wnt ligands regulate developmental processes individually and in combination. The findings may provide further insights into the role of Wnt signalling in developmental and degenerative diseases, including various types of cancer.