Deciphering terminal selector functions in motor neurons: from worms to humans - PROJECT SUMMARY/ABSTRACT: Loss of motor neuron (MN) function leads to debilitating diseases like amyotrophic lateral sclerosis (ALS). Victims of such diseases suffer from a lack of effective treatments in part due to our poor understanding of the molecular mechanisms that enable MNs to terminal differentiate and maintain function throughout life. The discovery of terminal selectors represented a pivotal advance in our understanding of these mechanisms. Terminal selectors are a class of transcription factors that establish and maintain neuronal terminal identity by directly activating expression of terminal identity genes encoding proteins that define the functional properties of neurons. Mutations in terminal selectors are implicated in severe neurodevelopmental disorders. Our understanding of terminal selector mechanisms remains limited by two main challenges: (1) they have only been studied using biased approaches, and (2) potential non-cell autonomous functions for terminal selectors are unknown. To overcome these two challenges, this proposal focuses on UNC-3 (Collier/Olf/Ebf), which functions as a terminal selector in C. elegans cholinergic MNs, and which is linked to a neurodevelopmental syndrome through mutations in its human ortholog, EBF3. Our recent ChIP-Seq study predicted > 3,500 genes as putative direct UNC-3 targets, only a fraction of which (2%) have been experimentally validated. To identify the transcriptional targets of UNC-3 in an unbiased manner, we performed single-cell RNA sequencing (scRNA-Seq) in adult wild- type and unc-3 mutants. This approach revealed thousands of up- and downregulated terminal identity genes in cholinergic MNs, suggesting a dual activator-repressor role for UNC-3 (Aim #1). Because our scRNA-seq strategy broadly profiled MNs in the mutant, we also uncovered massive transcriptional disruptions in GABA MNs, which do not express unc-3, suggesting that UNC-3 has non-cell autonomous functions. To uncover the mechanism through which UNC-3 affects GABA MN development non-cell autonomously, I will validate these effects in vivo and conducting a suppressor genetic screen (Aim #2). It is vital to extend insights from C. elegans to human MNs, as human UNC-3 ortholog EBF3 is linked to a neurodevelopmental syndrome. To do this, we employed human embryonic stem cell (hESC) technology. My preliminary data show that EBF1 and EBF3 are expressed in human ESC-derived MNs, but their function remains unknown. Based on our C. elegans studies, I hypothesize that EBFs controls terminal identity genes in human MNs. To test this, I will use small hairpin RNA (shRNA) to knockdown the expression of each human EBF gene in hESC-derived MNs paired with scRNA-Seq (Aim #3). Completion of this proposal will provide extensive training opportunities and valuable insights into gene regulatory mechanisms underlying MN terminal differentiation, which may aid the development of in vitro protocols to generate MNs and advance our molecular understanding of the EBF3 Syndrome and other neurodegenerative diseases.
