Despite our progress characterizing the molecular mechanisms by which post-mitotic neurons adopt their terminal identity features, it remains unclear if there are any overarching themes in regulation of terminal nervous system specification. In this grant proposal, I aim to analyze the function of a family of candidate regulators throughout an entire nervous system. To do this, I make use of the relatively simple C. elegans nervous system, which consists of 302 neurons in 118 anatomically distinct neurons classes. The mechanisms of neuronal identity specification have been probed in C. elegans, where genetic mutant analysis has uncovered a host of transcriptional regulators, designated terminal selectors. These terminal selectors control neuron-specific features by binding to cis regulatory regions upstream of neuron-specific genes and regulating their transcription. Of the terminal selectors that have been identified, most are homeodomain (HD) transcription factors belonging to the homeobox gene family. The broad usage of homeobox genes suggests that HD transcription factors may have evolutionarily ancient roles in specifying the terminal identity of an ancestral neuron type and, thus, I hypothesize that HD transcription factors are required for specification of every neuron in the C. elegans nervous system. To test this hypothesis, I propose analyzing the expression of more than 50 homeobox genes at a single neuron resolution using reporter strains. Then, I aim to determine if those HD transcription factors act as terminal selectors in the adult nervous system by examining the effect of their mutation on adoption of neurotransmitter identity. Thus far, I have completed examination of 22 homeobox reporters and found 20 to be expressed in post-mitotic neurons of the adult nervous system. One of those genes, ceh-34, was found to be a terminal selector in all the neurons where it is expressed. These results suggest that the proposal will likely identify novel regulators of terminal identity specification, and elucidate broad themes in development of post-mitotic neurons.
In this proposal, I aim to analyze the function of candidate, homeodomain-containing regulators throughout an entire nervous system, using C. elegans as a model. The successful completion of this project will create a map of regulatory molecules involved in specification of an entire nervous system, elucidating any overarching themes in development of post-mitotic neurons. Since the homeobox gene family has highly conserved functions across phyla, understanding their role in nervous system maintenance could contribute to genetic therapies for homeobox gene-related human diseases.
