This project will address the core mechanism underlying oscillatory Wnt signaling activity. Oscillatory Wnt signaling activity has so far been identified during mouse embryo segmentation, as several Wnt signaling target genes, such as Axin2, are periodically expressed (period ~2 hours). These oscillations occur within cells of the presomitic mesoderm (PSM), which are the undifferentiated precursors of somites, the prevertebrae. Despite an increasing list of oscillating genes, the fundamental mechanism how oscillations in Wnt pathway activity are generated in the first place, is still unknown. In this project, we will combine realtime quantification of signaling dynamics in mouse embryos with targeted functional perturbations in order to identify the mechanism underlying oscillatory Wnt signaling output. This project builds on recent achievements in my laboratory. First, we have developed an imaging reporter system enabling quantification of oscillatory signaling activities in real-time and directly within the developmental in vivo context. To this end, we have generated novel knock-in mouse lines that express highly dynamic fluorescent proteins or protein fusions, driven from endogenous loci of selected candidate Wnt target genes, such as Axin2. In addition, we established an in vitro segmentation assay that recapitulates mesoderm patterning and segmentation, including oscillatory pathway activities, in a highly simplified spatial 2-dimensional (2-D) context, enabling unprecedented experimental possibilities.Building on this experimental framework and in combination with functional perturbations (mouse genetics and small molecule approaches), we will investigate the mechanism generating oscillatory Wnt signaling activity. We will first address the role of negative feedback loops, specifically the role of the destruction complex that targets β-catenin for degradation, in generating and controlling oscillatory activity. Second, using an explorative screening approach combining siRNA knock-down that we recently established using the in vitro assay and real-time imaging, we aim to reveal novel regulators controlling temporal dynamics, i.e. oscillations, of Wnt signaling activity. Within the framework of SFB1324 and building on the mechanistic insight gained in this project, we will explore the implication of this fundamental, dynamic signaling property during mesoderm segmentation, but, also in other developmental context and in disease states.