Extracellular regulation of Chordin in a cnidarian

Bone Morphogenetic Protein (BMP) signaling is a critical means of cell-cell communication during animal development. In classical bilaterian model organisms (fruit flies and frogs), an interplay of the secreted BMP signals and their secreted regulator Chordin is required to coordinate the formation of tissues along the back-belly axis (known as the second axis) of the developing embryo. Interestingly, BMP and Chordin not only instruct the formation of the second body axis in animals belonging to the Bilateria (the group comprising most animal species), but also in some animals of a group called Cnidaria, the evolutionary sister group of Bilateria. Cnidarian Anthozoa (corals and sea anemones) have a BMP signaling-dependent second body axis, whereas cnidarian Medusozoa (jellyfish and hydroids) do not. Studying BMP signaling in Anthozoa is important to understand how BMP signaling-dependent second body axis formation evolved and learn about its potential function in the last common ancestor of Bilateria and Cnidaria. The present project investigates the function of the BMP regulator Chordin in the model anthozoan Nematostella vectensis, a sea anemone. Previous research has shown that the BMP-Chordin interplay during axial patterning in Nematostella is surprisingly similar to that in flies and frogs. While Chordin usually inhibits BMP signals, it can also promote BMP function, probably by enabling BMP transport. This so-called Chordin-mediated shuttling of BMP signals is crucial for axial patterning, yet we do not know how Chordin is transported extracellularly to perform its function as a shuttle. Moreover, it is unknown whether cleavage of the Chordin protein by enzymes called metalloproteases, which is critical to regulate Chordin in Bilateria, is important in Nematostella. To shed light on the extracellular regulation of Chordin in Nematostella and reveal the mechanisms behind Chordin function, the three main aims of the project are: 1) to visualize extracellular Chordin, 2) to identify extracellular regulators mediating Chordin transport, and 3) to characterize the possible function of metalloproteases in Chordin cleavage. Cutting-edge techniques will be used to achieve these goals. Transgenic sea anemones allowing highly efficient labeling of Chordin will be generated and used to visualize extracellular Chordin. To understand the mechanisms underlying extracellular Chordin transport, biochemical experiments will be combined with mass spectrometry to discover the extracellular protein binding partners of Chordin. Moreover, the potential cleavage sites in Nematostella Chordin will be determined and the importance of metalloproteases for Chordin cleavage will be tested by targeted disruption of their function. The present research project represents the first in-depth analysis of Chordin regulation outside of Bilateria. The results will provide important insights into the evolution of Chordin function and BMP signaling-mediated secondary body axes.