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  1. Author Response:

    Reviewer #1 (Public Review):

    In this tour-de-force analysis of transcriptional regulation and cell fate specification in C. elegans, Vidal et al. explore the role of the Sine Oculis/Six1/Six2 ortholog ceh-34 in the pharyngeal nervous system. Previous work demonstrated that ceh-34 is exclusively expressed in all pharyngeal neuron types. The current work data shows that ceh-34 function is required for the diverse differentiated features of all pharyngeal neuron subtypes, as well as their interconnectivity, but, interestingly, not for their basal "pan-neuronal" features. ceh-34 is also required to maintain these features, at least into larval stages. Convincing evidence is presented indicating that subtype-specificity emerges through the cooperation of ceh-34 with various individual homeodomain factors, consistent with the homeodomain "code" model that has emerged from this group's earlier work. One of the most fascinating aspects of this study is the association of ceh-34 with circuit formation, as it marks an entire set of interconnected neurons, and appears to be required for at least the gross features of this connectivity. The findings of Vidal et al. raise interesting questions about whether ceh-34 expression would be sufficient to endow non-pharyngeal neurons with pharyngeal characteristics, or whether it would instruct adjacent neurons and/or processes to form synaptic connections; these issues are not addressed in this work. There is also the potential for some confusion around nomenclature: the authors refer to the pharyngeal nervous system as the "enteric nervous system," which is not standard terminology in C. elegans. Previous work in the field has used "enteric" to describe muscles and neurons that regulate intestinal contraction and defecation; these are associated with the posterior intestine, not the pharynx. Nevertheless, the idea that the pharyngeal nervous system might share molecular similarities, and perhaps ancestry, with enteric circuits in other organisms is an interesting proposition. This speculation places the authors' findings into an evolutionary context that suggests a key role for homeodomain transcription factors in the specification of the enteric nervous system, proposing that more complex nervous systems may have evolved simple structures like the C. elegans pharyngeal nervous system.

    It does not seem that this reviewer is opposed to our usage of the term enteric neurons (which, by the way, has a precedent in a recent paper by the Flavell lab; PMID 30580965). We would nevertheless like to lay out our reasoning for this nomenclature. First, to refer specifically to what the reviewer says: the AVL and DVB neurons have indeed been called – justifiably so – “enteric neurons” before. What those neurons do is to innervate the HINDGUT, not more, not less. By the same token, neurons that innervate the FOREGUT deserve to be called enteric neurons as well – as they actually are across animal phylogeny. But irrespective of the hindgut/AVL/DVB case, the enteric nomenclature is clearly justified for the pharyngeal neurons because: (a) As per any animal anatomical textbook definition, the enteric nervous system is the nervous system of the gastrointestinal system. (b) Again, as per any textbook definition, the foregut is part of the gastrointestinal system. (c) The pharynx is the worm foregut and hence, the nervous system of the pharynx constitutes an enteric nervous system. (The foregut terminology for the worm pharynx has been used extensively before in the literature and for a good reason: Most animals, including mammals and humans, have a pharynx, which is considered part of the foregut. The only “unusual” thing about worms is that its pharynx is the only part of the foregut, while in other animals (incl. us), the foregut contains the pharynx, plus additional subdivisions, like the esophagus). (d) As importantly, the classification of the pharyngeal nervous system as an enteric system is also underscored by functional criteria: The two most distinguishing features of an enteric nervous system in animals, namely (i) its autonomous function and (ii) its rhythmic control of peristaltic movement are the defining features of the pharyngeal nervous system as well. We have carefully considered this matter and have also double-checked this terminology issue with the world expert of animal enteric nervous systems, our colleague Mike Gershon, who authored the book “The second brain” (= the enteric nervous system). In the revised version, we clarify this definitional issue in the Introduction.

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  2. Evaluation Summary:

    This paper will be of interest to developmental neurobiologists working on the transcriptional control of neural cell fate and connectivity. The data largely support the authors' finding that a single homeodomain transcription factor is a circuit-wide specifier of cell fate that functions combinatorially with other factors in the development of the C. elegans pharyngeal nervous system. The authors speculate about intriguing similarities between the nematode pharyngeal nervous system and vertebrate enteric nervous systems.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their name with the authors.)

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  3. Reviewer #1 (Public Review):

    In this tour-de-force analysis of transcriptional regulation and cell fate specification in C. elegans, Vidal et al. explore the role of the Sine Oculis/Six1/Six2 ortholog ceh-34 in the pharyngeal nervous system. Previous work demonstrated that ceh-34 is exclusively expressed in all pharyngeal neuron types. The current work data shows that ceh-34 function is required for the diverse differentiated features of all pharyngeal neuron subtypes, as well as their interconnectivity, but, interestingly, not for their basal "pan-neuronal" features. ceh-34 is also required to maintain these features, at least into larval stages. Convincing evidence is presented indicating that subtype-specificity emerges through the cooperation of ceh-34 with various individual homeodomain factors, consistent with the homeodomain "code" model that has emerged from this group's earlier work. One of the most fascinating aspects of this study is the association of ceh-34 with circuit formation, as it marks an entire set of interconnected neurons, and appears to be required for at least the gross features of this connectivity. The findings of Vidal et al. raise interesting questions about whether ceh-34 expression would be sufficient to endow non-pharyngeal neurons with pharyngeal characteristics, or whether it would instruct adjacent neurons and/or processes to form synaptic connections; these issues are not addressed in this work. There is also the potential for some confusion around nomenclature: the authors refer to the pharyngeal nervous system as the "enteric nervous system," which is not standard terminology in C. elegans. Previous work in the field has used "enteric" to describe muscles and neurons that regulate intestinal contraction and defecation; these are associated with the posterior intestine, not the pharynx. Nevertheless, the idea that the pharyngeal nervous system might share molecular similarities, and perhaps ancestry, with enteric circuits in other organisms is an interesting proposition. This speculation places the authors' findings into an evolutionary context that suggests a key role for homeodomain transcription factors in the specification of the enteric nervous system, proposing that more complex nervous systems may have evolved simple structures like the C. elegans pharyngeal nervous system.

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  4. Reviewer #2 (Public Review):

    Vidal et al. describe the role of the SIX-family transcription factor CEH-34 in the development of the pharyngeal nervous system. The manuscript is strong in several ways. The study is comprehensive and elegantly presented. The authors' conclusions are, with only a few minor exceptions, clearly supported by the presented data. And the study uses sophisticated genetic approaches to determine gene expression in vivo and manipulate gene function.

    The manuscript has some weaknesses. First, some of the manuscript is redundant with published data. The authors could better consolidate and synthesize what is known about the pharyngeal nervous system and CEH-34. The authors could also relegate data that support a point already made to supplementary figures. Second, the study does not address functional consequences of disrupting CEH-34 function. The pharynx is an excellent model for the genetic study of neural circuits and how they are assembled not only because of its defined anatomy but also because its function is readily assessed. Finally, the overarching premise of the study is that the pharyngeal nervous system is a model for the enteric nervous system, and that this model can be used to illuminate developmental mechanisms that have heretofore been poorly understood. This premise causes some confusion. The pharynx is not gut, and calling the pharyngeal nervous system the 'enteric' nervous system might raise an eyebrow. Also, the authors do not compellingly demonstrate that the development of the pharyngeal nervous system follows rules that differ from those that govern development of the rest of the nervous system. Any conceptual distinction that the authors attempt to make in their introduction is not relevant to their major conclusions.

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  5. Reviewer #3 (Public Review):

    This study addresses the development of the enteric nervous system in C. elegans. The authors find that the homeobox gene ceh-34 is specifically expressed in all 14 classes of pharyngeal neurons, but no other neurons in the animal. Mutant analysis reveals that ceh-34 is necessary for the differentiation of all 14 neuron classes. ceh-34 acts with several other homeodomain TFs to specify the different neuron classes. The authors speculate that the selective role for ceh-34 in specifying the entire enteric nervous system supports the notion of this system as perhaps the primordial origin of the nervous system.

    The study is overall of high quality and the conclusions balanced. While the combinatorial coding of neuronal subtype cell fate by TFs dates back at least two decades, this concept has not hitherto been probed in the enteric nervous system, which makes the study particularly interesting.

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