The development and function of individual neurons are defined by their unique transcriptomic properties, but despite recent efforts cataloguing single neuron transcriptomes, there remains a gap in our understanding of the causal mechanisms by which gene regulatory factors specify individual neuronal transcriptomes. In particular, little is known about how factors regulating various layers of gene expression, e.g. transcription factors (TFs) and RNA binding proteins (RBPs), coordinately control the transcriptomes of single neurons. This proposal aims to fill the gap by leveraging unique properties of the nematode Caenorhabditis elegans to mechanistically investigate coordinated transcriptomic regulation of specific model neurons in vivo. The well-described and invariant lineage of the C. elegans nervous system, combined with powerful genetic techniques, will enable detailed dissection of TF-RBP control over neuronal development. Additional tools recently developed and adapted in the lab, including combinatorial CRISPR/Cas9, single-neuron in vivo alternative splicing reporters, and neuron-specific FACS sorting followed by RNA Seq, will reveal mechanisms and consequences of coordinated regulation of single neurons in vivo. The objective of this proposal is to define TF-RBP pairs that genetically interact and combinatorially shape neuron-specific transcriptomes. The hypothesis is that cell-specific combinations of TFs and RBPs converge on specific target networks to define neuronal transcriptomes. This hypothesis is supported by preliminary in vivo data in C. elegans showing that (a) certain TFs and RBPs combinatorially define splicing choices including splicing of the conserved neuronal kinase sad-1 in individual neurons such as the touch-sensing neurons, and (b) neuronal TFs and RBPs genetically interact to affect neuronal function and behavior. The hypothesis will be further tested by the experiments proposed in the following aims: 1) Determine molecular mechanisms by which the neuronal TFs and RBPs we have identified coordinately control sad-1 alternative splicing in touch neurons, 2) Define functional consequences of dysregulated touch neuron transcriptomes when these regulatory factors or their target transcripts are lost, and 3) Systematically identify neuronal TFs and RBPs coordinately controlling neuron fate and function in specific tractable neuronal cell types. The expected outcomes of the proposed work are to determine mechanisms and functional consequences of coordinate TF-RBP control over single neuron transcriptomes. The proposed approach is innovative as it departs from the status quo by examining causal mechanisms and consequences of single-neuron transcriptomic regulation across multiple layers of gene regulation in vivo. It is significant because it is expected to advance the field of single-neuron transcriptomics into causal mechanisms, functional consequences, and coordinated regulation in single neurons in vivo. Ultimately, these findings will inform our understanding of how nervous systems develop and are specified.
The research proposed here is relevant to the mission of the NIH because understanding how networks of gene expression are generated in individual neurons is critical to understanding how these neurons arise and function, and understanding how individual neurons arise and function is critical to understanding normal and aberrant function of the human nervous system.