Sea star oocytes as a model system for Wnt pathway activation through novel signaling organelles - Project Summary Biochemical communication between cells is essential for many processes in early development, including the establishment of body axes and the specification of germ layers during gastrulation. The Wnt pathway is a conserved regulator of these events, and its dysregulation can lead to defects in gastrulation, nervous system development, and other processes. Mutations in Wnt pathway components are drivers of cancer in adults. The integration of Wnt ligands requires an enigmatic protein called Dishevelled (Dvl), which upon Wnt binding to its receptor, assembles into cytoplasmic puncta across species. These structures are hypothesized to activate the downstream pathway, but their composition, physical nature, and function are poorly defined. Adding further complexity, Wnt ligands are biochemically limited in their ability to diffuse. This constrains the distances at which cells can signal. To overcome this, some cells use long projections that can extend and touch other cells, such as cytonemes and nanotubes. However, these structures are diverse and challenging to study because their small size for microscopy, and lack of ways to selectively perturb them. We have developed the sea star oocyte system as a generalizable cellular test tube for Wnt signaling with unique advantages over other established models. We recently found that upon hormonal stimulation of these cells, Dvl synchronously assembles into large cytoplasmic assemblies at side of oocytes that will become the embryonic posterior. What are they made of, how do they assemble, and what is their function? In my independent lab, we have also discovered a population of microtubule-based projections that emanate from the Dvl region of the oocyte (oocyte-derived microtubule projections, OMPs). We hypothesize that these structures allow the cell to receive distant Wnt signals required for Dvl assembly formation. For this proposal, we will undertake the following themes: First, we will define the structure and function of Dvl assemblies. We will combine expansion and super-resolution light microscopy, in vivo structure-function approaches, and quantitative mass spectrometry to determine the physical and biochemical nature of Dvl assemblies. Second, we will determine the structure and function of OMPs. We will use live imaging and developmental approaches in ovaries and early embryos. This work will bring us toward our long-term goal of understanding the biochemical and biophysical logic of Dvl granule assembly. It will also define the structure and function of OMPs, which to our knowledge, have not been previously identified in any other oocyte system. This knowledge will clarify longstanding questions on the cellular regulation of Wnt signaling, with important implications for early development, regeneration, and cancer.
