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

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 C. elegans that resides within the foregut (pharynx) of the worm. A Caenorhabditis 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, without affecting panneuronal features. 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.


INTRODUCTION
Across animal phylogeny, the enteric nervous system constitutes a self-contained, autonomously acting neuronal network that detects physiological conditions to control the peristaltic movement of food through the digestive tract (Ayali, 2004;Copenhaver, 2007;Fung and Vanden Berghe, 2020;Hartenstein, 1997;Laranjeira and Pachnis, 2009). Because of structural and functional autonomy, the enteric nervous system of humans has been referred to as a "second brain" (Gershon, 1998). In mammals, the enteric nervous system is composed of around 20 different neuron types, categorized into internal sensory, inter-or motor neurons, which line the interior lumen of different sections of the digestive system (Drokhlyansky et al., 2020;Furness, 2000). While a lot of progress has been made in understanding early developmental patterning events that establish the fate of neurons in the enteric nervous system of mammals (Nagy and Goldstein, 2017;Sasselli et al., 2012), fish (Ganz, 2018) and flies (Copenhaver, 2007;Myers et al., 2018), very little is known about the later steps of enteric neurons terminal differentiation, both in vertebrate and invertebrate models (Copenhaver, 2007;Hao and Young, 2009;Memic et al., 2018;Morarach et al., 2021;Rao and Gershon, 2018). Specifically, it has remained unclear as to whether there are common unifying themes in how enteric neurons acquire their terminally differentiated state. This is particularly interesting from an evolutionary standpoint. The function of enteric neurons in controlling feeding behavior is an ancient one that may precede the evolution of the bilaterian central nervous system Furness and Stebbing, 2018;Gilbert, 2019;Koizumi, 2007). Understanding how enteric neurons acquire their terminal features may therefore provide novel insights into nervous system evolution.
The nematode C. elegans contains an enteric nervous system in its foregut, the pharynx (Albertson and Thomson, 1976;Mango, 2007). The pharyngeal nervous system shares features of enteric nervous systems of more complex animals. It is an autonomously acting unit, composed of 20 synaptically interconnected neurons that fall into 14 anatomically distinct classes, required for movement of food through the digestive tract of the worm (Fig.1) (Albertson and Thomson, 1976;Avery, 2012;Mango, 2007). Like other enteric nervous systems, the nematode pharyngeal nervous system is a non-centralized neuronal network isolated from the rest of the nervous system and, in rough analogy to the vagus nerve, is connected to the remainder of the nervous system through a single nerve fiber, that of the bilateral RIP neuron pair (Albertson and Thomson, 1976;Cook et al., 2019;White et al., 1986). Like vertebrate enteric neurons (Fung and Vanden Berghe, 2020), pharyngeal neurons have sensory, inter-and motorneuron function (Avery and Horvitz, 1989;Trojanowski et al., 2014). Our recent re-analysis of pharyngeal nervous system anatomy has shown that rather than segregating these functions over distinct neurons as vertebrate do (Fung and Vanden Berghe, 2020), most pharyngeal neurons each combine sensory, inter-and motorneuron function, i.e. are polymodal . The 14 distinct types of pharyngeal neurons are defined by their unique anatomy, i.e. axonal projections, morphology, synaptic connectivity ( Fig.1B,C)  and unique functional features (Avery and Horvitz, 1989;Trojanowski et al., 2014). This anatomical and functional classification has recently been further extended by the description of their unique molecular fingerprint, determined by scRNA transcriptomic analysis  (Fig.1D). For example, each pharyngeal neuron class is uniquely defined by characteristic signatures of neurotransmitter systems, from acetylcholine (ACh), glutamate (Glu) to serotonin, and by unique combinations of neuropeptide-encoding genes (Fig.1E). While pharyngeal neurons are clearly different from one another, scRNA profiling has shown that their molecular signatures are more similar to each other than to other neurons in the nervous system  (Fig.1D).
One fascinating aspect of the C. elegans pharyngeal nervous system is that it has an appearance of what one could imagine an ancestral, primitive nervous system to have looked like. Primitive nervous systems are generally thought to have emerged out of the context of monolayers of epithelial cells, with individual cells in such layers specializing into primitive sensory-motor-type neurons (Arendt, 2008;Mackie, 1970). These diffusely organized neurons may have sensed the environment and relayed such sensory information to the other primitive cell type thought to have arisen early in evolution, namely, contractile "myoepithelial" cells that were able to generate motion. The organization of the C. elegans pharynx reveals some striking parallels to such a presumptive primitive nervous system: It is also essentially a single monolayer of cells that are organized into a tubular structure and the vast majority of constituent cells are myoepithial cells (pharyngeal muscle) and polymodal, interconnected sensory/motor neurons Mango, 2007;Portereiko and Mango, 2001). Pharyngeal neurons combine sensory, inter-and motor neuron features and are also not localized to ganglia but rather diffusely localized, resembling the architecture of more ancient nerve nets (Albertson and Thomson, 1976;Watanabe et al., 2009). The 4 interconnectivity of pharyngeal neurons displays also less selectivity that non-pharyngeal neurons, such that mere physical proximity is an almost sufficient criteria for connectivity . Moreover, pharyngeal neurons are closely related to non-neuronal pharyngeal cells by lineage; for example, some muscle and neurons derive from a single mother cells (Sulston et al., 1983). The idea of enteric neurons being reflective of an early, primitive state of the nervous system has also been brought forward in the context of comparing enteric nervous systems from widely divergent species (Furness and Stebbing, 2018;Gilbert, 2019). Specifically, these authors argued that the nerve net-like hydra nervous system displays features of the vertebrate enteric nervous system.
The self-contained and hypothetically primitive state of the C. elegans enteric nervous system, with all its unique features, encouraged us to use this system as a model to probe several concepts of neuronal identity specification that have emerged from the centralized, non-pharyngeal nervous system of C. elegans: (1) The first is the concept of terminal selectors, transcription factors that act in a master-regulatory manner to coordinately control the many identity features of a terminally differentiating neuron (Hobert, 2016). Are members of terminal gene batteries in each pharyngeal neuron also controlled in a coordinated manner, via terminal selectors? One study in the NSM neurons provided some limited evidence in this regard , but how broadly this applies throughout the pharyngeal nervous system was less clear. (2) Second, homeodomain transcription factors have a predominant role as terminal selectors of neuronal identity in the non-pharyngeal nervous system (Hobert, 2021;Reilly et al., 2020). A recent cataloguing of the expression patterns of all homeodomain proteins in the C. elegans genome revealed that each one of the C. elegans 118 neuron classes, including the pharyngeal neurons, display a unique combinatorial signature of homeobox gene expression (Reilly et al., 2020). Are all pharyngeal neurons indeed specified by homeobox gene combinations(s)? (3) Lastly, there is evidence from both C. elegans (Berghoff et al., 2021;Pereira et al., 2015) and other systems (Brunet and Pattyn, 2002), that synaptically connected neurons are often specified by the same transcription factor, suggesting that such transcription factors may be involved in assembling neurons in functional circuitry. We have termed such transcription factors "circuit organizers" (Berghoff et al., 2021;Pereira et al., 2015) and sought to test whether the isolated pharyngeal circuitry is similarly specified by a circuit organizer transcription factor.

5
In this paper, we show that all three predictions are fulfilled in the context of the nematode's pharyngeal/enteric nervous system. We show that a C. elegans ortholog of the Sine Oculis/Six1/Six2 homeobox gene, ceh-34, is expressed in all pharyngeal neurons from their birth throughout their life time. Its expression is induced by the foregut organ selector gene pha-4/FoxA. We demonstrate that ceh-34 initiates and maintains the terminally differentiated state of all synaptically connected pharyngeal neurons, that ceh-34 is required to assemble pharyngeal neurons into proper circuitry and that in distinct pharyngeal neuron types, ceh-34 cooperates with distinct homeobox genes to specify their respective identity.
Taken together, our studies further substantiate overarching themes of nervous system development and potentially provide insights into the evolution of nervous systems.

Expression of paralogous genes ceh-34 and ceh-33, the two C. elegans Sine
Oculis/Six1/2 orthologs The first systematic analysis of developmental patterning genes conducted after the release of the C. elegans genome sequence revealed several C. elegans homologs of the Sine oculis/Six family of homeodomain proteins (Ruvkun and Hobert, 1998) whose founding member was first identified in Drosophila for its role in eye patterning (Cheyette et al., 1994).
This specific homeodomain transcription factor family is characterized by the presence of a conserved domain, located N-terminally to the DNA binding homeodomain, the ~150 amino acid long SIX domain, involved in both protein-DNA, as well as protein-protein interactions (Kumar, 2009;Patrick et al., 2013). C. elegans Six-type homeodomain proteins fall into several, phylogenetically conserved families, the Sine Oculis/Six1/2, the Six4/5 and the Six3/6 subfamily (Dozier et al., 2001;Kumar, 2009). We focus here on the Sine Oculis subfamily.
Through the analysis of multiple nematode genome sequences we found that nematodes generally contain a single ortholog of the Sine Oculis/Six1/2 subfamily of SIX homeodomain proteins, but in the Caenorhabditis genus this locus has duplicated into two immediately adjacent paralogs, ceh-33 and ceh-34 ( Fig.2A, Fig.2 -Supplement 1). Using a fosmid-based reporter in which the ceh-33 locus is tagged with gfp, we find that the CEH-33 protein shows no expression in the nervous system within or outside the pharynx at any developmental stage. The only observed expression was in a subset of head muscle cells ( Fig.2B).
A previously described reporter construct that contains the coding region and 3.8kb of promoter region of the neighboring ceh-34 gene showed expression in all pharyngeal neurons, but no other neurons outside the pharynx . A fosmid-based reporter shows the same expression pattern (Reilly et al., 2020). To further confirm this strikingly selective pattern of neuronal expression and also to examine expression at different developmental stages with the best possible reagent, we used the CRISPR/Cas9 system to engineer a reporter allele of ceh-34. This reporter shows the same expression as the fosmidbased reporter in all pharyngeal neurons, but no other neurons (Fig.2B). The ceh-34 reporter 7 is turned on in the embryo at around the time of birth of pharyngeal neurons and is maintained in all pharyngeal neurons throughout all larval and adult stages (Fig.2).
The remarkable restriction of ceh-34 expression within the nervous system to all pharyngeal neurons prompted us to ask whether the Forkhead transcription factor pha-4, an organ selector gene involved in early patterning of the pharynx (Gaudet and Mango, 2002;Horner et al., 1998;Kalb et al., 1998;, is required for ceh-34 expression. Crossing a ceh-34 reporter into pha-4(q490) mutant animals, we indeed observed a loss of ceh-34 expression (Fig.2). Conversely, ceh-34 does not affect pha-4 expression ( Fig.2 -Supplement 2A). The regulation of ceh-34 by pha-4 mirrors the effects that pha-4 has on the expression of other transcription factors that control terminal differentiation of other tissue types in the pharynx, for example the ceh-22/NKX homeobox gene that specifies pharyngeal muscle differentiation (Vilimas et al., 2004), or the hlh-6 bHLH gene that specifies pharyngeal gland differentiation (Smit et al., 2008). We furthermore note that a pha-4 reporter allele that we generated through CRISPR/Cas9 genome engineering is continuously expressed throughout the entire pharyngeal nervous system, at all postembryonic stages ( Fig.2 -Supplement 2B), raising the possibility that pha-4 not may only initiate, but also maintain ceh-34 expression.

ceh-34 controls the expression of diverse neurotransmitter signaling pathways in pharyngeal neurons
To begin to assess the function of ceh-34 in enteric nervous system differentiation, we generated a null allele of the ceh-34 locus, ot1014, using CRISPR/Cas9 engineering, in which the entire ceh-34 locus is deleted ( Fig.2A). Previously isolated alleles include a hypmorphic missense allele and a small deletion allele, tm3733 . In our ensuing mutant analysis, we found ot1014 and the previously described strong loss of function allele tm3733 are phenotypically indistinguishable and we therefore used both alleles interchangeably.
We first asked whether ceh-34 is required for the generation of pharyngeal neurons.
We next assessed the effect of ceh-34 on a large collection of neuron type-specific molecular identity features (illustrated in Fig.1E). To this end, we first focused on the signaling capacities of all neurons. In the vertebrate enteric nervous system, each individual neuron class is distinguished by its unique set of signaling molecules, from classic neurotransmitters to neuropeptides (Morarach et al., 2021). The same applies to all neuron classes in the pharyngeal/enteric nervous system of C. elegans (Horvitz et al., 1982;Pereira et al., 2015;Taylor et al., 2021). We first considered the three classic neurotransmitters ACh, Glu and serotonin which are employed in C. elegans much like in the enteric nervous system of other species: Seven of the 14 pharyngeal neuron classes use acetylcholine as neurotransmitter, four use glutamate and one uses serotonin (NSM) (Horvitz et al., 1982;Pereira et al., 2015;Taylor et al., 2021). Of those neurotransmitters, serotonin is perhaps the best studied neurotransmitter both in the vertebrate enteric nervous system (Gershon, 2013) as well as in the nematode pharyngeal nervous system (Horvitz et al., 1982;Ishita et al., 2020;Song and Avery, 2013).
Acquisition of ACh, Glu and serotonin neurotransmitter identity features can be visualized through the expression of a number of enzymes and transporters: unc-17/VAChT for cholinergic identity, eat-4/VGluT for glutamatergic identity and tph-1/TPH, cat-1/VMAT, bas-1/AAAD and cat-4/GCH for serotonergic identity. We examined the expression of all these markers, using various reporter genes, in all pharyngeal neurons of ceh-34 mutant animals and found that the seven cholinergic, four glutamatergic and single serotonergic neuron classes fail to acquire their respective neurotransmitter identity (Fig.3B). We schematize these results in the context of a pharyngeal circuit diagram to illustrate the breadth of effects of ceh-34 on neurotransmitter identity acquisition (Fig.3C).
As in vertebrate enteric nervous systems (Drokhlyansky et al., 2020), pharyngeal neurons also display highly patterned expression of various neurotransmitter receptors . We analyzed the ceh-34-dependence of three exemplary receptors, the serotonin receptor ser-7, which mediates several functions of pharyngeal serotonin (Hobson et al., 2006), as well as a metabotropic and an ionotropic Glu receptor, mgl-1 and glr-2, each expressed in specific subsets of pharyngeal neurons. We find the expression of these 9 receptors in different pharyngeal neuron types is strongly affected, if not entirely abrogated upon removal of ceh-34 (Fig.4).

ceh-34 controls diverse neuropeptidergic identities of pharyngeal neurons
The function of vertebrate enteric nervous system is modulated by a number of prominent neuropeptidergic signaling systems (Abot et al., 2018;Llewellyn-Smith, 1989). In some cases, both neuropeptide and receptor are expressed in the vertebrate enteric nervous system, while in others, the peptide is produced elsewhere but acts on neuropeptide receptors located in the enteric nervous system. Likewise, the C. elegans pharyngeal nervous system expresses a great diversity of neuropeptides and neuropeptide receptors, including homologs of neuropeptide signaling systems that function in the vertebrate enteric nervous system . In fact, each pharyngeal neuron expresses a unique combination of neuropeptides and their receptor proteins  (Fig.5). We tested whether ceh-34 affects the neuron-type specific expression of various neuropeptidergic signaling systems.
We first examined the expression of the phylogenetically conserved Thyrotropinreleasing hormone (TRH) signaling axis that is important in stimulated gastrointestinal motility in vertebrates (Abot et al., 2018). C. elegans homologs of either the Thyrotropin-releasing hormone (TRH-1) or its receptor (TRHR-1) are expressed in the pharyngeal nervous system (Hunt-Newbury et al., 2007;Van Sinay et al., 2017), a notion we confirmed and extended with CRISPR/Cas9-engineered reporter alleles, showing that trh-1 is expressed in the MI, M4 and M5 neurons and trhr-1 in the M1, M2, M3 and I5 neurons (Fig.4, Fig.5). We find that the expression of the trh-1 and trhr-1 reporter alleles in these pharyngeal neurons is strongly affected in ceh-34 mutants (Fig.4, Fig.5).
In addition to this deeply conserved neuropeptidergic system, we also tested the expression of a cohort of neuropeptides from the FMRFamides (flp-2, flp-4, flp-5, flp-15, flp-21, flp-28) and other miscellaneous neuropeptides (nlp-3, nlp-8, nlp-13). The expression of these nine neuropeptides is neuron-type specific, but in aggregate they cover the entire pharyngeal nervous system, often with unique cell-type specific combinations (schematized in Fig.5B). We find that the expression of all of these nine neuropeptides, analyzed with either reporter transgenes or CRISPR/Cas9 genome-engineered reporter alleles, is affected in ceh-34 mutants (Fig.5A). We schematize these results again in the context of a pharyngeal circuit diagram to illustrate the breadth of effects of ceh-34 on neuropeptide expression (Fig.5B). Together with our analysis of classic neurotransmitters (ACh, Glu, serotonin) and receptors, we conclude that ceh-34 is required to endow pharyngeal neurons with their neuron type-specific arsenal of signaling molecules and, hence, that ceh-34 is a critical specifier of several key aspects of pharyngeal neuron identity.

ceh-34 is required for sensory receptor expression in the pharyngeal nervous system
We sought to extend our analysis of ceh-34 mutants by examining the expression of other molecular features of pharyngeal neurons. Like neurons in the vertebrate enteric nervous system, many of the pharyngeal neurons are likely sensory neurons that perceive sensory information to modulate peristaltic movements of the alimentary tract . While the sensory apparatus of pharyngeal neurons is not well understood, there are several candidate sensory receptors expressed in pharyngeal neurons. A gustatory receptor family member, gur-3, a possible light receptor (Bhatla and Horvitz, 2015), is expressed in two pharyngeal neuron classes and its expression is lost in ceh-34 mutants (Fig.6A).
Pharyngeal neurons also express the two sole members of the ionotropic sensory receptor family (Croset et al., 2010), encoded by glr-7 and glr-8 in C. elegans Hobert, 2013). We examined expression of glr-7 expression, normally observed in six pharyngeal neuron classes (I2, I3, I6, M2, M3 and NSM), in ceh-34 mutants and found its expression to be completely lost (Fig.6A). Finally we analyzed the expression of str-97, a putative chemosensory receptor of the GPCR family which we found to be expressed in several pharyngeal neurons   (Fig.6A). We found that ceh-34 is required for proper str-97 expression (Fig.6A).

ceh-34 is required for the expression of antimicrobial defense machinery
One deeply conserved feature of the gut and its associated nervous system is their engagement in antimicrobial defense, either directly through the release of antimicrobial peptides or through the employment of signaling systems that activate the immune system (Klimovich and Bosch, 2018;Muniz et al., 2012). Similar defense strategies operate in C.
elegans (Dierking et al., 2016). Aside from the epithelial cells of the intestines, the pharyngeal nervous system appears to play a direct role in these microbial control mechanisms, as inferred by the pharyngeal neuron expression of specific proteins implicated in antimicrobial defense. For example, pharyngeal neurons express a hormone, FLR-2, homologous to glycoprotein hormone alpha subunit, that signals to the intestine to orchestrate antimicrobial defense (Oishi et al., 2009). Pharyngeal neurons also secrete pore-forming polypeptides that directly kill bacteria, such as the SPP-12 protein (Hoeckendorf et al., 2012), as well as a defensin-type antimicrobial peptide, ABF-2 and fungal-induced peptides (FIPR proteins) (Kato et al., 2002;Taylor et al., 2021). Our scRNA transcriptome analysis  revealed pharyngeal neuron expression of another saposin-related secreted protein, which we named htrl-1 (see Methods). We visualized expression of these signaling molecules, using a promoter fusion for spp-12 (Hoeckendorf et al., 2012) and CRISPR/Cas9-engineered reporter alleles for flr-2 and htrl-1 (Fig.6B). We find that the spp-12 reporter is expressed in I4 and M4 neurons, the flr-2::SL2::gfp::h2b reporter allele is expressed in 10 of the 14 pharyngeal neuron classes and the htrl-1::SL2::gfp::h2b reporter allele is expressed in all pharyngeal neurons (and in all other pharyngeal cells, but nowhere outside the pharynx) (Fig.6B). A notable feature of the extrapharyngeal expression of the flr-2 reporter allele is expression in the AVL and DVB neurons (Fig.6B), the only extrapharyngeal neurons of the C. elegans nervous system that innervate gut tissue (not the foregut, but the midgut) (White et al., 1986). Crossing these reporters into a ceh-34 mutant background, we found that expression of all three genes (spp-12, flr-2, htrl-1) is severely reduced and/or eliminated in pharyngeal neurons (Fig.6B).

ceh-34 is required for the expression of pan-pharyngeal nervous system genes
In addition to investigating the ceh-34-dependence of genes that fall into specific functional categories, we also sought to capitalize on the recently released single cell transcriptome profiling of the entire C. elegans nervous system that included the entire pharyngeal nervous system . This analysis had shown that the molecular signatures of pharyngeal neurons are more similar to each other than to other neurons in the nervous system  (Fig.1D). This pattern is driven, in part, by: (a) the pan-pharyngeal neuron, but pharynx-exclusive expression of ceh-34, as well as its transcriptional cofactor, Eyes absent/eya-1 (described in more detail below), (b) pharynx restricted expression of the serotonin receptor ser-7, and several neuropeptides (e.g. tkr-1), all ceh-34 targets, as described above, (c) a number of previously entirely uncharacterized genes with very broad, if not panpharyngeal nervous system expression (but no expression in non-pharyngeal neurons), including the above-mentioned saposin-related htrl-1 gene, small cell surface proteins (e.g. C54E4.4) and a novel receptor tyrosine kinase with distant homology to the insulin receptor (Panther domain PTHR24416:SF525a), which we named kin-36 ( Fig.6 -Supplement 1). To confirm this expression pattern, we tagged the kin-36 locus with a gfp::H2B::SL2 cassette at it's 5' end, using CRISPR/Cas9 genome engineering. We found that gfp::kin-36 indeed displays pan-pharyngeal neuron expression; expression is also observed in all other pharyngeal cell types, but no cell types outside the pharynx (Fig.6C). This pattern is strikingly similar to that of the saposin-related htrl-1 reporter allele ( Fig.6B), making htrl-1 and kin-36 the only known genes whose expression is so exclusively coupled to all pharyngeal cell types, but no other cell type. Crossing gfp::kin-36 into ceh-34 mutants, we observe what appears to be selective loss of kin-36 expression from many, albeit not all pharyngeal neurons, a similar effect to what we observed for htrl-1 (Fig.6B, C).
We conclude that ceh-34 is required for the adoption of a broad palette of individual molecular features of all pharyngeal neurons (Table 1), consistent with a role as a terminal selector of neuronal identity of all pharyngeal neurons. Like other terminal selectors, ceh-34 only affects neuron-type specific features, but not features that are expressed by all neurons throughout the nervous system.

ceh-34 is continuously required to maintain the differentiated state
The effect of ceh-34 on terminal marker expression and its continuous expression throughout the life of all pharyngeal neurons suggests that, like other terminal selectors in the non-pharyngeal nervous system, ceh-34 may not only initiate but also maintain the terminally differentiated state. To test this possibility, we generated a conditional ceh-34 allele that allowed us to deplete CEH-34 protein postdevelopmentally. To this end, we inserted an auxin-inducible degron  into the ceh-34 locus using CRISPR/Cas9 genome engineering. Together with a ubiquitously expressed TIR1 ubiquitin ligase that recognizes the degron (eft-3 driver; ieSi57) , this approach allows for 13 temporal depletion of CEH-34 protein through addition of auxin to the plate. (Fig.7A) Constitutive auxin exposure does not phenocopy the larval arrest phenotype of ceh-34 null mutants, consistent with the persistence of very low level of CEH-34::mNG::AID (Fig.7).
Nevertheless, postembryonic auxin addition resulted in downregulation of two tested markers for the differentiated state of different pharyngeal neuron classes, eat-4/VgluT and unc-17/VAChT (Fig.7). We conclude that ceh-34 is required to maintain differentiated features of pharyngeal neurons and therefore fulfills another key criterion to classify as a terminal selector of pharyngeal neuron identity.

Pharyngeal nervous system architecture is severely disorganized in ceh-34 mutants
We further extended our analysis of ceh-34 function by analyzing the anatomy of pharyngeal neuron circuitry in ceh-34 mutants. This analysis is particularly important in light of the observation that, both in C. elegans (Berghoff et al., 2021;Pereira et al., 2015) and in vertebrates (Brunet and Pattyn, 2002), several instances have been described in which synaptically interconnected, but otherwise distinct neurons express the same transcription factor. Such observation suggests that these transcription factors may have a role in assembling neurons into functional circuitry. ceh-34 represents a particularly extreme version of this scenario, because all ceh-34(+) neurons are heavily synaptically interconnected and only make a single robust synaptic contact to the rest of the C. elegans nervous system ( Fig.1C) . To assess whether ceh-34 not only specifies terminal molecular properties of a neuron, but also organizes overall circuit architecture, we examined pharyngeal nervous system architecture using fluorescent reporter constructs. Such analysis is complicated by the fact that genes that are selectively expressed in ceh-34(+) neurons, and hence could serve as drivers for a fluorescent reporter, are turned off in ceh-34 null mutant animals, thereby preventing an easy visualization of individual axonal tracts or synaptic contacts. However, we found that the ceh-34 promoter itself is still expressed until the first larval stage in ceh-34 null mutants, when these animals arrest development. This transgene (otIs762) reveals that although their identity is not properly specified, as described above, pharyngeal neurons retain the capability to grow neuronal projections in ceh-34 mutants; however, axonal tracts are severely disorganized (Fig.8A).
We also expressed the cytoplasmically localized TagRFP reporter together with a 14 synaptically localized, GFP-tagged CLA-1/Clarinet protein (a synaptic active zone marker) (Xuan et al., 2017) under control of the ceh-34 promoter. In wild-type animals, this transgene (otIs785) reveals (a) the axonal tracts of the pharyngeal nervous system and (b) synaptic structures that are strongly enriched in the pharyngeal nerve ring (Fig.8B). In ceh-34 null mutants, we observed not only a disruption of axonal tract anatomy, but a severe disorganization of synaptic clusters throughout the entire pharyngeal nervous system ( Fig.8B).
In ceh-34(n4796) animals, the expression of many molecular markers for individual pharyngeal neurons are not affected, allowing to visualize their morphology. Focusing on two neuron types, NSM and I5, we find that in both types, specific axons branches fail to form in ceh-34(n4796) animals (Fig.8C). We conclude that ceh-34 is required to establish proper pharyngeal nervous system architecture.

ceh-34 affects the expression of molecules involved in proper wiring of the pharyngeal nervous system
To further explore how ceh-34 may affect pharyngeal nervous system architecture, we considered the expression of molecules with potential or explicitly demonstrated roles in axon guidance and/or synapse formation. A genetic analysis of axon guidance and circuit formation has only been conducted in a small number of pharyngeal neurons, mainly the M2 and NSM neuron classes (Pilon, 2008). In both neuron classes, the SLT-1 axon guidance cue, the C. elegans orthologue of Slit, has been found to be required for proper M2 and NSM axon guidance (Axang et al., 2008;Rauthan et al., 2007). We extended this phenotypic characterization, finding that other pharyngeal neurons also display axon pathfinding defects in slt-1 mutants (Fig.8 -Supplement 1). To examine potential links between slt-1 and ceh-34, we made use of a promoter::gfp fusion that captures the entire upstream intergenic region of the slt-1 locus  and which shows selective expression in seven pharyngeal neuron classes (I2, I6, M1, M2, M4, M5 and MI) at the first larval stage. We find that slt-1 expression is essentially abrogated in ceh-34 mutants (Fig.8D).
Effects of ceh-34 on molecules potentially involved in circuit formation are not restricted to slt-1. Two Ig superfamily members, rig-3 and rig-6 (the sole C. elegans ortholog of contactin) have previously been implicated in axon outgrowth and synapse function in the C. elegans nervous ssytem (Babu et al., 2011;Bhardwaj et al., 2020;Katidou et al., 2013;Kim and Emmons, 2017) and promoter fusion transgenes have indicated their expression in pharyngeal neurons (Schwarz et al., 2009). We used CRISPR/Cas9 to tag both loci with gfp and found that both genes are broadly expressed in many pharyngeal neurons (Fig.8E). ceh-34 affects the pharyngeal neuron expression of both of rig-3 and rig-6 reporter alleles (Fig.8E).

The Six homeodomain cofactor, Eyes absent/Eya, cooperates with ceh-34
To gain further insights into how CEH-34 patterns the identity of a wide array of distinct pharyngeal neuron types, we considered the involvement of cell-type specific cofactors. As a first step, we considered the EYes Absent/EYA protein, a phylogenetically conserved transcriptional co-activator of specific subsets of Six homeodomain proteins (Ohto et al., 1999;Patrick et al., 2013;Tadjuidje and Hegde, 2013). The C. elegans ortholog of Eyes absent, called eya-1 , also directly physically interacts with CEH-34 protein . We first examined eya-1 expression in pharyngeal neurons using a genomic fragment that contains the entire, gfp-tagged eya-1 locus . We observed expression in all pharyngeal neurons throughout all larval and adult stages, a precise phenocopy of the ceh-34 expression pattern (Fig.9A), an expression pattern also corroborated by our recent scRNA analysis ( Fig.6 -Supplement 1) . Moreover, we found that eya-1 expression requires ceh-34 function, suggesting that ceh-34 acts in a feedforward configuration to induce its own transcriptional cofactor (Fig.9B).
We analyzed the function of eya-1 in the context of pharyngeal neuron specification.
Animals that carry a deletion of a part of the eya-1 locus, ok654, display pharyngeal neuron specification defects, albeit much milder than those observed in ceh-34 null mutant animals ( Fig.9C). The larval arrest phenotype of ceh-34 null mutants is also more penetrant than that of eya-1 mutants, which are homozygous viable . Since it is not clear whether the eya-1 allele is a null allele, it remains unclear whether ceh-34 may be able to partly function without eya-1.
In other organisms, Dachshund proteins are components of Sine oculis/Eya complexes in several cellular contexts (Hanson, 2001). However, the sole C. elegans ortholog of Dachshund, dac-1, does not cooperate with CEH-34/EYA-1 in the context of pharynx development. First, dac-1 is not expressed in pharyngeal neurons (Colosimo et al., 2004;Taylor et al., 2021) and, second, dac-1 null mutants do not display the larval arrest phenotype characteristic of ceh-34 and eya-1 mutants (Colosimo et al., 2004).

ceh-34 cooperates with a multitude of other homeobox genes to specify distinct pharyngeal neuron types
How does ceh-34 activate distinct genes in different pharyngeal neuron types? One obvious possibility is that ceh-34 cooperates with neuron type-specific cofactors in neurontype specific terminal selector complexes to drive specific fates. As candidates for such cofactors, we considered homeobox genes, for two reasons: (1) like any other neuron in the C. elegans nervous system, each individual pharyngeal neuron expresses a unique combination of homeobox genes, in addition to pan-pharyngeal ceh-34 (Reilly et al., 2020); (2) previous studies had already implicated a few homeobox genes in controlling some select functional or molecular aspects of individual pharyngeal neurons Feng and Hope, 2013;Morck et al., 2004;Ramakrishnan and Okkema, 2014;Ray et al., 2008;. For example, the homeobox gene ceh-2, the C. elegans ortholog of vertebrate EMX and Drosophila Ems, is required for proper function of the M3 neuron, but effects of ceh-2 on molecular aspects of M3 neuron differentiation had not been reported . We therefore set out to analyze homeobox gene function throughout the pharyngeal nervous system and to examine interaction of ceh-34 with other homeobox genes.
NSM neurons: To ask whether distinct pharyngeal neuron type-specific homeobox genes cooperate with ceh-34 in distinct neuronal cell types throughout the pharyngeal nervous system, we made use of a hypomorphic ceh-34 allele, n4796 .
Unlike the ceh-34 null allele, which displays highly if not completely penetrant effects on all NSM molecular markers ( Fig.3-6), the n4796 allele displays only subtle if any neuronal differentiation defects on its own (Fig.10A). However, when combined a mutant allele of unc-86, a POU homeobox gene that affects many but not all NSM marker genes  strong synergistic differentiation defects of NSM are observed (Fig.10A). This genetic interaction mirrors the synergistic effects of unc-86 and the LIM homeobox gene ttx-3, another regulator of NSM differentiation .
I2 neurons: Synergistic interactions are also observed in the glutamatergic I2 neuron pair. Like the I1 neurons, no identity regulators were previously known for this neuron class.
Our homeobox gene mapping project (Reilly et al., 2020) showed that the I2 neuron expresses the LIM homeobox gene ceh-14, the C. elegans ortholog of vertebrate Lhx3/4. No other pharyngeal neuron expresses ceh-14. While ceh-14 single null mutants show no effect on eat-4/VGLUT expression (the marker of glutamatergic identity of the I2 neurons), in combination with the ceh-34(n4796) hypomorphic allele a strong synergistic effect on glutamatergic identity acquisition is observed in I2 (Fig.10C). Another molecular marker for I2 identity, the neuropeptide nlp-8, is also synergistically regulated by ceh-34 and ceh-14 ( Fig.10C).
M3 neuron: Apart from expression in I3, the EMX ortholog ceh-2 is also expressed in the glutamatergic M3 neurons and is required for M3 function , but molecular correlates for this functional defect have not previously been identified. We find that in ceh-2 single mutants, expression of the eat-4/VGluT identity marker is affected in M3 (Fig.11B).

M5 neuron:
The cholinergic M5 neuron expresses, in addition to ceh-34, the Msh/Msx ortholog vab-15 (Reilly et al., 2020). Since only a hypomorphic allele of vab-15 was previously available (Du and Chalfie, 2001), we generated a molecular null allele, ot1136, through CRISPR/Cas9 genome engineering (Fig.11 -Supplement 1). We found a complete loss of expression of the cholinergic marker unc-17/VAChT expression, as well as of the neuropeptidergic marker, trh-1, in vab-15(ot1136) animals (Fig.11C). Complete losses of marker expression are not an indicator of failure of this neuron to be generated, since crossing a ceh-34 marker into vab-15 mutants revealed the presence and normal ceh-34 expression of M5 (Fig.11C).
MI neuron: In our previous genome-wide analysis of homeobox gene expression (Reilly et al., 2020), we had shown that the glutamatergic MI neuron expresses the sole worm ortholog of the Goosecoid homeobox gene, ceh-45. Embryonically, ceh-45 is expressed in multiple pharyngeal tissues (Ma et al., 2021), but its expression resolves to exclusive expression in the MI, as well as the I1 neuron (Reilly et al., 2020). ceh-45 had not previously been functionally uncharacterized. We examined ceh-45 function by generating a null allele using CRISPR/Cas9 genome engineering, ot1065 (Fig.11 -Supplement 1). Mirroring the ceh-34 defects, we found that glutamatergic identity specification of MI (as assessed by eat-4/VGLUT expression) is strongly affected in ceh-45 null mutant animals (Fig.11D). Similarly, expression of the neuropeptide trh-1 is also affected in MI (Fig.11D).
In conclusion, eight homeobox genes appear to collaborate with ceh-34 in eight of the 14 pharyngeal neuron classes to specify their proper identity ( Table 1). Since the remaining six classes also express specific combinations of homeobox genes (Reilly et al., 2020), we 19 anticipate that future analysis will likely reveal homeobox codes throughout the entire pharyngeal nervous system.

DISCUSSION
We have identified here common, overarching themes of enteric nervous system differentiation in the nematode C. elegans. A number of previous studies have identified transcription factors involved in regulating specific differentiation aspects of a small subset of pharyngeal neurons Feng and Hope, 2013;Morck et al., 2004;Ramakrishnan and Okkema, 2014;Rauthan et al., 2007;Ray et al., 2008;), yet no common theme emerged from these studies. We have shown here that a Sine oculis ortholog, ceh-34, operates together with its phylogenetically conserved transcriptional coactivator eya-1, to selectively specify the terminal differentiation program of all pharyngeal neurons. The ceh-34/eya-1 complex appears to act as a terminal selector of pharyngeal neuron identity, as inferred from its requirement to initiate terminal neuronal differentiation programs in all pharyngeal neurons (without overall loss of panneuronal identity, a unifying trait of all terminal selectors), as well as its continuous role in maintaining the differentiated state (another defining trait of terminal selectors). The aspects of the differentiation program that we consider here, and found to be under control of ceh-34, include anatomical (axon outgrowth and synapse formation), molecular and presumptive functional features. Molecular and functional features affected by ceh-34 range from neuron-neuron communication to presumptive sensory functions to the intriguing function of enteric neurons as potential regulators of microbial colonization. There is good reason to believe that CEH-34 controls these diverse phenotypic identity features in a direct manner, i.e. it may not act through intermediary factors. CEH-34 is among the many C. elegans transcription factors whose binding sites have been determined in vitro through protein binding microarrays (Narasimhan et al., 2015) and, using a phylogenetic footprinting pipeline, we find that these motifs are significantly enriched in the single cell transcriptome of most pharyngeal neuron classes . This is in accordance with CEH-34 being a shared terminal selector component of all pharyngeal neuron classes. Within the nervous system, the selectivity of ceh-34 expression in all pharyngeal neurons is remarkable -based on extensive gene expression pattern analysis, including recent scRNA data, there is no other transcription factor that so selectively and comprehensively defines all enteric, but no non-enteric neuronal cell types. We found that the key determinant of this expression is the organ selector PHA-4 (Gaudet and Mango, 2002;Horner et al., 1998;Kalb et al., 1998;, which is expressed earlier in development to act both as a pioneer factor (Hsu et al., 2015) and to induce the expression of a number of different terminal selectors for different tissue types within the foregut -the ceh-34 gene for all neurons (this paper), the bHLH transcription factor hlh-6 for pharyngeal gland cells (Smit et al., 2008) and the Nk-type homeobox gene ceh-22 for pharyngeal muscle identity (Vilimas et al., 2004). Given the continuous pharyngeal expression of PHA-4 throughout postembryonic life that we observe, it is conceivable that PHA-4 acts in a regulatory feedforward motif configuration, where it not only induces tissue-type terminal selectors (ceh-34, hlh-6, ceh-22), but then also collaborates with them to induce and maintain terminal differentiation batteries.
Our work indicates that CEH-34 is a shared component of neuron-type specific terminal selector complexes, such that CEH-34 interacts with a distinct set of at least eight homeodomain co-factors to impose unique features in distinct pharyngeal neuron types.
This in vitro-determined binding sites for these homeodomain co-factors display, like the CEH-34 site, a phylogenetically conserved enrichment in the respective neuron typespecific gene batteries . Taken together, our findings not only reveal a common terminal selector-based regulatory logic for how a self-contained, enteric nervous system acquires its terminal differentiated state. They also corroborate two themes that have emerged from recent studies, primarily in C. elegans (and also emerging in other systems): (1) homeobox genes -and more specifically, homeobox gene combinatorial codesare prominently employed in neuron identity specification throughout all neurons of the nervous system, as exemplified here in the context of the enteric nervous system, the socalled "second brain" of animals (Gershon, 1998).
(2) A number of identity-specifying terminal selectors, such as CEH-34, are expressed in synaptically connected neurons, suggesting they may specify the assembly of neurons into functional circuitry. It is presently unclear how prominent such a connectivity theme is. We observed a few cases in the non-pharyngeal nervous system (Berghoff et al., 22 2021;Pereira et al., 2015) and there are some striking examples in vertebrates (Brunet and Pattyn, 2002;Dauger et al., 2003;Ha and Dougherty, 2018;Ruiz-Reig et al., 2019;Sokolowski et al., 2015). This present study provides an extreme example of this -an entire set of synaptically connected neurons (the worm's enteric nervous system) is specified by a single transcription factor (CEH-34), which apparently helps these neurons in being assembled into functional circuitry. In each pharyngeal neuron type, CEH-34 pairs up with different homeodomain proteins to diversify pharyngeal neurons into distinct identities. Following Dobzhansky's dictum that "nothing in biology makes sense except in the light of evolution" (Dobzhansky, 1964) we speculate that the pharyngeal nervous system may have derived from a homogenous set of interconnected, identical neurons, all specified by ceh-34, which may have regulated a homophilic adhesion molecule that functionally linked these ancestral neurons. The more complex, present day circuitry may have evolved through the eventual partnering of CEH-34 protein with additional homeodomain proteins that diversified neuronal identities, connectivity and function in the pharyngeal nervous system.

Arguing for a conserved function of Six homeodomain factors in enteric nervous
system differentiation is the observation that in flies, the Sine oculis paralog Optix is indeed expressed in the frontal ganglion (Seo et al., 1999), which constitutes the nervous system of insect foreguts (Hartenstein, 1997). In the context of studying Sine Oculis function in the Drosophila corpus cardiacum, it was also noted that the entire stomatogastric ganglion, i.e. the entire enteric nervous system (of which the frontal ganglion is a part) does not form in Sine oculis mutants (De Velasco et al., 2004). In sea urchin, pharyngeal neurons also express, and require for their proper development, the Sine oculis paralog Six3 (Wei et al., 2011). Other than an early report of Six2 expression in the mouse foregut region (Ohto et al., 1998), the expression and function of Sine oculis orthologs in vertebrate enteric nervous systems has not yet been examined.
Notably, another homeobox gene appears to have a critical and very broad function in vertebrate enteric nervous system development that is akin to the broadness of ceh-34 function in C. elegans. The Prd-type homeobox gene Phox2b is expressed in enteric nervous system precursors and required early in development for generation of all enteric ganglia (Pattyn et al., 1999;Tiveron et al., 1996). While Phox2b is also continuously expressed throughout the adult enteric nervous system (Corpening et al., 2008;23 Drokhlyansky et al., 2020;Morarach et al., 2021), its function in terminal differentiation and perhaps even maintenance of enteric neurons identity remains to be examined, for example, via temporally controlled, post-developmental knock-out in juvenile or adult stage animals. If the analogy to ceh-34 holds, the enteric neurons of such animals may lose their differentiated state. Remarkably, additional homeobox genes have recently been noted to show highly selective expression patterns within the vertebrate enteric nervous system, effectively discriminating distinct neuronal subtypes (Memic et al., 2018). One of them, the Meis ortholog Pbx3 has been confirmed to play an important role in the postmitotic specification of distinct enteric neuron types (Morarach et al., 2021). Hence, it appears that the overall logic of a pan-enteric homeobox gene, cooperating with cell type specific homeobox genes, may be conserved from worms to vertebrates.
Another evolutionary perspective of our findings considers the origins of the enteric nervous system, and maybe nervous systems as a whole. Based on a number of anatomical and functional features, it has been proposed that enteric nervous systems preceded, and then paralleled the existence of centralized nervous system of bilaterian animals (Furness and Stebbing, 2018;Gilbert, 2019;Klimovich and Bosch, 2018). This argument is bolstered by considering a number of features of the enteric, i.e. pharyngeal nervous system of C. elegans: (a) its polymodality (sensory + inter + motor neuron) of most pharyngeal neurons, (b) its innervation of what is essentially a single sheath of myoepithelial cells, a proposed feature of primitive nervous systems (Mackie, 1970), (c) its simple immune functions (also thought to be a feature of primitive neurons, e.g. in hydra) (Klimovich et al., 2020;Klimovich and Bosch, 2018) and (d) the relatively indiscriminate synaptic crossinnervation patterns among pharyngeal neurons . If pharyngeal neurons indeed resemble a more primitive, ancestral state of neurons, our observation that CEH-34 acts as a terminal selector in these neurons would point to the ancient nature of (a) a terminal selector-type logic of neuronal identity specification and (b) the deployment of a homeobox gene in such function. Sine Oculis homologs appear to be employed broadly in sensory neuron specification across animal phylogeny, even in the most basal metazoan (Jacobs et al., 2007). CEH-34/Sine Oculis may represent an ancestral determinant of neuronal cell types.

Microscopy
Worms were anesthetized using 100 mM sodium azide (NaN3) and mounted on 5% agarose pads on glass slides. Z-stack images (each ~0.7 micron thick) were acquired using a Zeiss confocal microscope (LSM880) or Zeiss compound microscope (Imager Z2) using the ZEN software. Maximum intensity projections of 2 to 30 slices were generated with the ImageJ software (Schindelin et al., 2012).

Caenorhabditis elegans strains
Worms were grown at 20°C on nematode growth media (NGM) plates seeded with E.
coli (OP50) bacteria as a food source. The wild-type strain used is Bristol N2. A complete list of strains used in this study can be found in Supplemental File 1.
ceh-34(ot1188) and ceh-34(ot1189) were generated the same way as ceh-34(ot1014) and are molecularly identical. The difference is that ot1188 was generated in the background of flr-2(syb4861) and ot1189 was generated in the background of htrl-1(syb4895) because these loci are very closely linked.
The pha-4 locus was tagged with gfp at its 3'end to generate pha-4(ot946), using Cas9 protein, tracrRNA, and crRNA from IDT, as previously described (Dokshin et al., 2018). One crRNA (attggagatttataggttgg) and an asymmetric double stranded gfp-loxP-3xFLAG cassette, amplified from a plasmid, were used to insert the fluorescent tag at the C-terminal.

Generation of transgenic reporter strains
To generate otIs762(ceh-34prom::TagRFP) a 3720bp PCR fragment containing the whole ceh-34 intergenic region plus the first 2 exons and 2 introns was amplified from N2 genomic DNA and cloned into a TagRFP vector using Gibson Assembly (NEBuilder HiFi DNA Assembly Master Mix, Catalog # E2621L). The following primers were used: aatgaaataagcttgcatgcctgcaTGTTTATTTTCTATGTAATTTCTAATAAAGTCCC and cccggggatcctctagagtcgacctgcaCTGAAAGTTGAAATATAGAATTTTTAATTTTTTTTTTTTG. The resulting construct was injected as a simple extrachromosomal array (50ng/ul) into pha-1(e2123) animals, using a pha-1 rescuing plasmid (pBX, 50ng/ul) as co-injection marker. A representative line was integrated into the genome with gamma irradiation and backcrossed 4 times.
To generate otIs785(ceh-34prom::GFP::CLA-1) a 3720bp PCR fragment containing the whole ceh-34 intergenic region plus the first 2 exons and 2 introns was amplified from N2 genomic DNA and cloned into PK065 (kindly shared by Peri Kurshan) using Gibson Assembly (NEBuilder HiFi DNA Assembly Master Mix, Catalog # E2621L). The following primers were used: gattacgccaagcttgcatgcTGTTTATTTTCTATGTAATTTCTAATAAAGTC and gttcttctcctttactcatcccgggCTGAAAGTTGAAATATAGAATTTTTAATTTTTTTTTTTTG. The resulting construct was injected at 7ng/ul together with ceh-34prom::TagRFP (50ng/ul) (see above) and rol-6(su1006) as a co-injection marker. A representative line was integrated into the genome with gamma irradiation and backcrossed 4 times.

Choice of pharyngeal fate markers
Most fate markers used in this paper were previously described. For several of those, we used previous expression patterns (based on transgenic reporter fusions) as a guide to then generate reporter alleles by CRISPR/Cas9 genome engineering (e.g. flp-5, ser-7; all listed in previous sections). One case warrants specific emphasis: scRNA has shown that the T11F9.12 gene is expressed exclusively in most, if not all pharyngeal neurons, a notion we confirmed with a CRISPR/Cas9 genome engineered reporter allele. T11F9.12 encodes for a relatively large (736aa), secreted and nematode specific protein that contains a Pfamannoted domain (Htrl domain; PF09612) that is, outside nematodes, only found in a bacterial protein HtrL. This bacterial protein currently has no assigned function but is found in a region of LPS core biosynthesis genes which are involved in bacterial immune defense (Bertani and Ruiz, 2018). Interestingly, hidden Markov model-based searches in the Panther database reveal a sequence pattern (PTHR21579) along the entire T11F9.12 protein that is otherwise only found in C. elegans saposin proteins, which are bona fide immune effector proteins (Banyai and Patthy, 1998;Hoeckendorf et al., 2012;Oishi et al., 2009). We named this protein HTRL-1.

Temporally controlled CEH-34 protein degradation
We used conditional protein depletion with the auxin-inducible degradation system . AID-tagged proteins are conditionally degraded when exposed to auxin in the presence of TIR1. To generate the experimental strain, the conditional allele ceh- ) was crossed with ieSi57 [eft-3 prom ::tir1], which expresses TIR1 ubiquitously. The natural auxin indole-3-acetic acid (IAA) was purchased from Alfa Aesar 27 (Ward Hill, MA) (#A10556) and dissolved in ethanol (EtOH) to prepare 400-mM stock solutions. NGM agar plates with fully grown OP50 bacterial lawn were coated with the auxin stock solution to a final concentration of 4 mM and allowed to dry for a few hours at room temperature. To induce protein degradation, worms of the experimental strain were transferred onto the auxin-coated plates and kept at 20°C. As a control, worms were transferred onto EtOH-coated plates instead. Auxin solutions, auxin-coated plates, and experimental plates were shielded from light.  ceh-34 expression in head muscle cells, marked with red asterisk, is not affected since they do not express pha-4.      Statistical analysis was performed using unpaired t-test.