Defining motor neuron diversity from embryo to adulthood and generating tools for in vivo and in vitro access

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PROJECT SUMMARY/ABSTRACT In order to understand neurological diseases, it is essential to identify the affected neuronal cell types, create model systems that accurately recapitulate normal function and disease phenotypes, and develop tools that allow cellular manipulations. Motor neurons in the spinal cord control body movement by communicating central motor commands with muscle targets. All spinal motor neurons are born and specified during embryonic development, but their molecular identities and electrophysiological properties evolve for weeks in mice and months in humans, until motor circuits and behavior become fully mature in post-natal life. In diseases like Amyotrophic Lateral Sclerosis (ALS), sarcopenia, or spinal cord injury, the degeneration of specific subsets of mature, adult motor neurons can lead to loss of muscle control, paralysis, and death. Although several studies have mapped the mechanisms of motor neuron diversification in the embryonic spinal cord, our understanding of motor neuron diversity in the adult spinal cord is in its infancy. This hinders the study of adult motor neuron diseases as the affected motor neuron subtypes are not thoroughly defined. Furthermore, in vitro models that faithfully recapitulate adult motor neuron identity do not exist, and adequate tools to access specific motor neuron subtypes in vivo are lacking. This research plan aims to map the trajectory of post-mitotic motor neurons from embryo to adulthood and use this data to both create viral tools that provide genetic access to specific subtypes of motor neurons in vivo, and develop methods for generating adult-like motor neurons in vitro. This will be done by first performing single cell transcriptome and chromatin profiling in mouse spinal motor neurons at various embryonic to adult ages. The temporal chromatin profiles will be used to develop AAV tools that provide genetic access to specific motor neuron subtypes at all ages, and to computationally identify candidate regulators of subtype- and adult-specific identity. The identified regulators will then be used to mature the age of mouse stem cell derived motor neurons in vitro. Finally, single cell transcriptomic and chromatin accessibility profiles of adult human motor neurons will be generated and a combination of mouse and human-specific regulators will be used to program the age of iPSC-derived motor neurons. This thorough approach will define the molecular features of motor neuron subtypes that contribute to their differential susceptibility in disease, and establish tools necessary for dissecting circuits, disease modeling, and the delivery of potential therapeutics. The training phase of the award will be conducted in the laboratory of Dr. Hynek Wichterle at Columbia University, and under the co- mentorship of Dr. David Gifford and Dr. Paola Arlotta. My career development plan describes a detailed timeline for acquiring all the technical and professional skills necessary for the successful transition into a career as an independent researcher. The completion of the proposed research plan will facilitate future research in my lab aimed at understanding the temporal dynamics of neuronal identity, circuits, and disease in motor neurons and other nervous system cells.