Abstract: Wnt signaling is one of the most important pathways in metazoans, but it is not existent in fungi, plants, or other eukaryotes. We discovered that the pathway exists in the freshwater polyp Hydra and in other cnidarians (jellyfish), which are the sister group to bilaterian animals. Moreover, our work also revealed thatthese ancient creatures already exhibit the complete repertoire of Wnt ligands and antagonists normally found only in vertebrates. During development and regeneration Wnts are consecutively activated along the main body axis in a cascade-like manner, with a peak of Wnt activity at the site of a blastopore-like signaling center (organizer) of these gastrula-like organisms. The pattern of Wnt activation in cnidarians is highly reminiscent to the morphogen gradient of Wnt/β-catenin signaling that regulates anteroposterior neural patterning during vertebrate brain development and was characterized as the “Wnt code”. It remains unclear how the interplay of different Wnt ligands, receptors and co-receptors contributes to the patterning of these simple organisms. In order to understand the specific function of different Wnt ligands, a mechanistic knowledge of the receptor-ligand interactions in this paradigmatic model system is indispensable. We will use three main approaches: (i) We will interfere with the function of specific Wnts in vivo using either siRNA knock-down or CRISPR/Cas9 knock-out approaches that we have successfully established in the lab. (ii) We will analyze the specificity of different Fzd-Lrp5/6-combinations for the different Wnt-ligands in cell-based TOPFLASH Wnt reporter assays and cell-free biochemical ligand-receptor assays. (iii) Since the spatio-temporal expression pattern of different Wnt proteins as well as their diffusion range may specifically affect the functional relevance of the Wnt code, we will use transgenic animals expressing HA-tagged Wnts to monitor their diffusion range. We will also produce transgenic lines bearing Fzd-GFP fusion proteins to monitor the receptor dynamics. By this combined approach we will be able to unravel the molecular basis for the specificity of secreted Wnt ligands toward the Fzd receptors and Lrp5/6 co-receptor in Hydra, which will shed light on the evolution of the Wnt pathway.