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.
