Fundamental underpinnings of adult subtype diversification in spinal motor neurons

About This Grant

PROJECT SUMMARY Building a nervous system requires both the specification and maturation of diverse neuron types. Most post-mitotic neurons are specified during embryonic development, but their synaptic connections and electrophysiological properties continue to mature during postnatal life. Compared to our understanding of embryonic neuronal specification, the regulatory mechanisms that orchestrate functional maturation of neurons in postnatal life remain poorly understood. The goal of this proposal is to fill this gap in our understanding of skeletal motor neurons. Skeletal motor neurons innervate muscles throughout the body to control movement throughout life. To accomplish this, adult skeletal motor neurons are found in three subtypes: alpha, gamma, and type3/beta, which have different morphology, electrophysiological properties, and circuit components. Importantly, these subtypes also show differential susceptibility to degeneration in the lethal disease Amyotrophic Lateral Sclerosis (ALS): alpha motor neurons die, gammas survive, and the fate of type3/betas is not known. Despite the fact that embryonic motor neuron development has been intensely studied for many years now, we do not understand how these functionally important and disease relevant adult subtypes are generated. In this proposal, we aim to fill this gap by utilizing novel single nuclei sequencing datasets and subtype-specific AAV tools that we have generated. By performing single nuclei RNA- and ATAC-sequencing on mouse motor neurons from embryonic, neonatal, juvenile and adult stages, we have found that adult alpha, gamma, and type3 subtype identities are established during the process of maturation. We have also identified candidate regulators that include subtype-specific, activity-independent transcription factors, and shared, activity- dependent transcription factors. In Aim 1 of this proposal, we will delineate the temporal maturation trajectory of all three subtypes by performing RNA-FISH against temporally activated genes. We will also use subtype- specific enhancer-AAV reporters to determine when subtype-specific muscle innervation patterns are established. In Aim 2, we will functionally interrogate the role of subtype-specific transcription factors in controlling mature gene expression, muscle innervation patterns, and motor behaviors. In Aim 3, we will functionally test the role of neural activity in controlling maturation of all subtypes. We will also map the binding profiles of subtype-specific and activity-dependent transcription factors to understand how they work together to generate mature and functionally distinct subtype identities. This work will lead to a ground-breaking understanding of adult motor neuron development, identify molecular and functional differences between disease relevant cell types, and inform methods to generate adult-like motor neurons from stem cells. The conceptual advances and AAV tools generated by this study will lead to improvements in treating diseases like ALS and spinal cord injury.