JAK/STAT signalling in vertebrates is activated by multiple cytokines and growth factors. By contrast, the Drosophila genome encodes for only three related JAK/STAT ligands, Upd, Upd2 and Upd3. Identifying the differences between these three ligands will ultimately lead to a greater understanding of this disease-related signalling pathway and its roles in development. Here, we describe the analysis of the least well characterised of the Upd-like ligands, Upd3. We show that in tissue culture-based assays Upd3-GFP is secreted from cells and appears to interact with the extracellular matrix (ECM) in a similar manner to Upd, while still non-autonomously activating JAK/STAT signalling. Quantification of each of the Upd-like ligands in conditioned media has allowed us to determine the activity of equal amounts of each ligand on JAK/STAT ex vivo and reveals that Upd is the most potent ligand in this system. Finally, investigations into the effects of ectopic expression of Upd3 in vivo have confirmed its ability to activate pathway signalling at long-distance.
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