Systematic generation of <i>Drosophila</i> Wnt transgenes enables the characterization of canonical Wnt signaling

A conserved cohort of signalling pathways orchestrate development and adult homoeostasis. Deregulation of these pathways underlies many diseases. A key set of signals is the family of Wnt ligands. Members of this family are conserved, but a clear understanding of the unique and redundant roles is lacking. Previous efforts to study Wnt ligand function in <i>Drosophila</i> have been hampered by the difficulty of generating , functional transgenes. To address this, we have created a complete set of synthesized constructs in an insulated expression system, integrated into the same genomic location, enabling reliable gain-of-function analyses across multiple tissues. Distinct phenotypic outcomes were observed, reflecting both shared and unique features of individual ligands. To define the canonicity of Wnt signalling, we monitored canonical targets such as <i>Dfz3, notum</i>, and the <i>'naked cuticle'</i> phenotype in developing tissues and the adult gut. Our findings revealed strong evidence of canonical responses from not only <i>wg</i>, but also <i>DWnt6</i> and <i>DWnt10</i> in the embryo, wing disc, larval gut, and adult gut. In addition, <i>DWnt2, DWnt4</i>, and <i>WntD</i> produced phenotypes distinct from the control with <i>DWnt2</i> and <i>DWnt4</i>, showing context-dependent evidence of some canonical activity. While previous studies have suggested regulatory features between <i>wg</i> and <i>DWnt6</i>, our work provides functional evidence that Wg, DWnt6, and DWnt10 each induce expression of canonical signalling reporters <i>in vivo</i>. These findings refine our understanding of redundancy and specificity within the <i>Drosophila</i> Wnt family and demonstrate that multiple Wnt ligands can act similarly within the canonical pathway depending on tissue context.