The enteric nervous system of the C. elegans pharynx is specified by the Sine oculis-like homeobox gene ceh-34

Abstract

Overarching themes in the terminal differentiation of the enteric nervous system, an autonomously acting unit of animal nervous systems, have so far eluded discovery. We describe here the overall regulatory logic of enteric nervous system differentiation of the nematode Caenorhabditis elegans that resides within the foregut (pharynx) of the worm. A C. elegans homolog of the Drosophila Sine oculis homeobox gene, ceh-34, is expressed in all 14 classes of interconnected pharyngeal neurons from their birth throughout their life time, but in no other neuron type of the entire animal. Constitutive and temporally controlled ceh-34 removal shows that ceh-34 is required to initiate and maintain the neuron type-specific terminal differentiation program of all pharyngeal neuron classes, including their circuit assembly. Through additional genetic loss of function analysis, we show that within each pharyngeal neuron class, ceh-34 cooperates with different homeodomain transcription factors to individuate distinct pharyngeal neuron classes. Our analysis underscores the critical role of homeobox genes in neuronal identity specification and links them to the control of neuronal circuit assembly of the enteric nervous system. Together with the pharyngeal nervous system simplicity as well as its specification by a Sine oculis homolog, our findings invite speculations about the early evolution of nervous systems.

Widespread employment of conserved C. elegans homeobox genes in neuronal identity specification

“…For example, the set of neurons that co-express the homeodomain protein CEH-34 and the set of neurons that co-express the homeodomain protein UNC-42 share more molecular similarities among themselves than with other neuron classes (Fig.1B). This is particularly notable because both the UNC-42(+) and CEH-34(+) neurons are synaptically more interconnected than expected by chance (Berghoff et al, 2021; Vidal et al, 2022).…”

“…However, absences of defects are not straight-forward to interpret for multiple reasons: First, because our cell fate marker analysis only usually tested a small number of markers, one cannot exclude that other markers would show a defect in expression. Second, in several previously documented cases, even unrelated homeobox genes from different subfamilies (e.g., LIM, POU) can act redundantly in neuron identity specification (Vidal et al, 2022; Zhang et al, 2014). For example, neither ttx-3 nor unc-86 single mutants affect expression of several different differentiation markers of the NSM neurons, but in a ttx-3; unc-86 double mutants these markers are completely lost (Zhang et al, 2014).…”

“…1B). This is particularly notable because both the UNC-42(+) and neurons are synaptically more interconnected than expected by chance (Berghoff et al, 2021;Vidal et al, 2022).…”

“…We have engaged in such holistic analysis in the nematode C. elegans , which contains a nervous system with substantial cellular diversity but limited overall number: 302 neurons in the hermaphrodite which fall into 118 classes (White et al, 1986). In our search for common principles in C. elegans neuron type specification, several themes have emerged: (1) the direct, coordinated control of neuron type specific gene batteries by so-called “terminal selectors” (Glenwinkel et al, 2021; Hobert, 2008, 2016b); (2) the overrepresentation of homeodomain transcription factors as terminal selectors (Hobert, 2021; Reilly et al, 2020) and (3) the codification of individual neuron identities by distinct combinations of homeodomain proteins, unique for each individual neuron type (Hobert, 2021; Reilly et al, 2020; Vidal et al, 2022).…”

“…Fig.S4: eya-1 null mutant does not affect expression of AIA differentiation markers. We introduced the same CRISPR null deletion as in(Vidal 2022) in the unc-17 and flp-19 reporter alleles syb4491 and syb3278, yielding alleles ot1208 and ot1209 . A small Zstack (6-7 images at .8 micron) around the middle of the worm are shown with 10 uM scale bars.…”