Neuronal specification by homeodomain transcription factors in Caenorhabditis elegans

The goal of this project was to elucidate the role of the homeodomain transcription factor family in terminal fate specification of an entire nervous system. In pursuit of this, I systematically identified the expression patterns of all 102 homeobox genes in the L4/adult stage C. elegans nervous system at a single-neuron resolution. This involved acquiring and/or generating high-quality fluorescent reporter reagents to tag all 102 homeodomain transcription factor proteins. Then, analyzing the expression of those reagents using a novel tool for whole nervous system identification in C. elegans, called NeuroPAL. The resulting expression atlas is the first complete picture of the homeobox family in any nervous system. It allowed the identification of new terminal selector proteins, including ceh-8, ceh-32, and ceh-31, in various neuron types and will continue to serve as a guide for future terminal selector identification across the nervous system. We discovered that every neuron type, and many subtypes, of the C. elegans nervous system express a completely unique set of homeodomain transcription factors. This unique expression code, along with the scores of homeodomain terminal selectors, suggests the possibility that every C. elegans neuron type is specified by a homeodomain terminal selector. We further probed the importance of the homeobox family in neuron specification by comparing its expression pattern with other transcription factor families. This necessitated high-quality data for the other major transcription factor families, bHLH, ZF, AtHook, bZip, and NHR, in every C. elegans neuron type. In collaboration with the labs of David Miller III at Vanderbilt and Marc Hammarlund at Yale, we used single cell RNA sequencing (scRNA-Seq) to molecularly profile all neuron types of the L4 stage C. elegans nervous system. We found that our homeodomain protein atlas was recapitulated fairly well in the scRNA-Seq data when thresholded and determined that the homeodomain transcription factor family is not alone in generating unique expression profiles for every neuron type. Two larger transcription factor families, ZF and NHR, are also uniquely expressed in each neuron identity. Instead, we found that the homeodomain transcription factor family is set apart from other families by their distinctly sparse expression across the nervous system at comparatively high levels. These expression patterns along with the numerous examples of functional homeodomain terminal selectors suggest that the family is an underlying theme in neuronal specification. We further extended this analysis to available scRNA-Seq datasets in the mouse nervous system and noted select commonalities in homeobox family expression across organisms. In all, this study shows yet again that analyzing homeodomain transcription factors leads to fruitful insights on organismal development. We found that the complexity of the C. elegans nervous system can be categorized and largely specified by a single family of transcription factors, building on previous studies of their importance in neuronal function.