Collaborative Research: Evolution of ligand-dependent Robo receptor activation mechanisms for axon guidance

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The connections between the information-processing cells of our nervous system are formed by long extensions of these cells called axons. The growth and guidance of axons toward their correct target cells is a critical step in neural circuit assembly during embryonic development. One important group of proteins that reside on growing axons and control their guidance is called Robos. This collaborative study will elucidate the molecular mechanisms underlying the activation of Robo family proteins for axon steering, and it will also determine how Robos have changed over the course of evolution to allow the wiring of nervous systems with increasing complexity. By providing insights into the molecular mechanisms of axon pathfinding, this work can improve our understanding of disorders resulting from nervous system miswiring, and it has the potential to inform therapeutic approaches for neural circuit repair. The project will involve undergraduate and graduate students in cutting-edge research, with an emphasis on the career development of women and underrepresented minorities. It also includes outreach to Providence, RI, and Chicago, IL, public high schools by hosting students in research laboratories and helping them gain early experience in developmental neurobiology research.The Slit-Robo ligand-receptor pair is an evolutionarily ancient signaling module that controls axon crossing of the nervous system midline in bilaterians by mediating repulsion. Yet, our understanding of the signaling mechanisms of Robo and Slit homologs and other associated proteins remains incomplete. Further complicating the picture is a divergent Robo family member, mammalian Robo3, which cannot bind Slits but is essential for axon crossing of the spinal cord midline. Previous work discovered a secreted Robo3 ligand, NELL2, which guides commissural axons via repulsion. Preliminary results indicate that the formation of an active NELL2-Robo3 complex is mechanistically distinct from Slit-Robo interactions and that, in mammals, Robo3 and the canonical Robos, Robo1, and Robo2, evolved from a dual ligand-binding ancestor at the root of the vertebrate tree. The driving hypothesis for the proposed studies is that the functional specialization of mammalian Robo family members is crucial for circuit wiring. This project integrates the expertise of two investigators in structural/biophysical and functional/genetic approaches to investigate Robo signaling and evolution. Ancient sequence reconstruction, biochemical methods, and axon guidance assays will be used to map the evolutionary history of Robo ligand binding and signaling capabilities. Further, structural approaches, structure-based mutagenesis, biophysical methods, and axon guidance assays will be combined to determine the contributions of Robo ligand binding site divergence, extracellular domain conformation, and molecular crowding to ligand-receptor complex formation and signaling in mammals and non-mammalian vertebrates. The project will provide training to students at all levels and include outreach activities, with a research focus, at local high schools.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.