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Abstract

The nervous systems of many nematodes are remarkably similar despite considerable divergence of their genomes. To examine nematode nervous system evolution, we are characterizing the nervous system of Pristionchus pacificus (Ppa) to compare with that of C. elegans (Cel) and other nematodes. To identify all neurons in Ppa, we have examined expression of the homeodomain TF gene ceh-48, which is known in Cel to be pan-neuronal. Using two independent strains in which the native CEH-48 protein is epitope- tagged with 2xFLAG, we have determined that Ppa-ceh-48 is likely also pan-neuronally expressed. We find that the number of ceh-48-expressing nuclei in the head of P. pacificus matches the number of neurons identified by EM reconstruction (both published and in preparation). For example, we can readily identify 13 neuronal nuclei in the anterior pharynx (defined by EM in Bumbarger et al., 2013, Cell 152: 109), and also appropriate numbers in the ganglia surrounding the nerve ring. We have used ceh-48 staining to determine the numbers of neuronal nuclei in the retrovesicular ganglion, ventral nerve cord, preanal and postanal (tail) ganglia, and the body wall, to define the full complement of neurons in P. pacificus. We have learned that compact nuclear morphology such as that seen with the DNA stain DAPI is not always a reliable indicator of a neuron: some ventral nerve cord neurons, for example, have a distinctly non-typical or ‘non-neuronal’ appearance to their nuclei. As a part of our nervous system characterization, we are also identifying neurotransmitters in Ppa by immunostaining with antibodies to GABA, serotonin, and cholinergic proteins. Lastly, we have also examined by epitope-tagging (FLAG and/or ALFA) expression patterns of several transcription factor (TF) genes likely mediating neuronal specification in Ppa, including Ppa orthologs of unc-3, unc-42, and unc-86 genes, known in C. elegans to act as ‘terminal selectors’ (e.g., Hobert, 2016; Curr Top Dev Biol 116: 455-475).