Context-Dependent, Combinatorial Logic of BMP Signaling

Evolution generated diverse signaling proteins for the control of multicellular patterns and organ- isms. These include the proteins of the Bone Morphogenetic Protein (BMP) pathway. Nearly a dozen BMPs activate the BMP pathway to promote the formation of tissues as diverse as bone, cartilage, blood vessels, and the kidney, making them attractive therapeutics for regenerating those tissues in adults. During development, the response to a given BMP depends heavily on context, such as which other BMPs are present and which BMP receptors are expressed on the cell being ac- tivated. However, despite knowing that context matters, the overall logic of this context-dependent signal processing, including the roles of specific ligands and receptors in shaping context and how this logic arises from biochemical features of specific pathway components, remains unclear. Inspired by maps of gene epistasis and drug interactions that functionally classify members of complex biological systems, we comprehensively measured responses to all pairs of ten BMP homodimers (BMP2, BMP4, BMP5, BMP6, BMP7, BMP9, BMP10, GDF5, GDF6, and GDF7), combining robotic liquid handling with a high-throughput fluorescent reporter of pathway activa- tion. These data functionally classify ligands into "equivalence groups," or ligands that combine in the same way with all other ligands across combinations. Surprisingly, the functional groupings do not correlate with similarity of ligand sequence and can change with cell context. Together, the context-dependent equivalence groups summarize the diverse responses to combinations of BMP ligands and their dependence on specific BMP receptors. The experimentally observed pairwise responses are also consistent with a mathematical model where BMP ligands compete for limited BMP receptors with different affinities and then produce outputs with different ligand-specific activ- ities. Ultimately, these results provide a useful reference for explaining the unique effects of BMP combinations in different tissues or time points in development, as well as highlighting counter- intuitive mechanisms for this complex signal processing. Chapter 1 provides an introduction to how and why we study cell-cell signaling. Chapter 2 provides a summary of the determination of equivalence groups, their dependence on receptor context, and fitting the mathematical model of receptor competition. Chapter 3 provides suggestions for future work, including recommendations for improved model fitting as well as crucial extensions to the definitions of BMP "combinations" and "context" to deepen our understanding and control of this critical pathway