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    Author Response to Reviewer Comments

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    Manuscript number: RC-2021-00979

    Corresponding author(s): Horvitz, H Robert

    Reviewer #1:

    *Major comments:

    The manuscript is very well written and results have been very clearly presented. The key conclusions drawn by the authors are convincing. However, one of the claims by the authors is not supported by the data. In lines 206-215 the authors discuss experiments where they visualized the morphology of the AIAs in ctbp-1 mutants where ctbp-1 expression is restored temporally in the L4-young adult stage using a heat-shock promoter construct. The authors conclude that "ctbp-1 can act ... in older worms to maintain aspects of AIA morphology in a manner similar to AIA gene expression." However, the data presented in Fig. 3I-L show no statistically significant difference between ctbp-1 mutants and mutants with the HS-construct, either with and without heat shock. Thus, although there seems to be some effect of the heat shock, this is not significant and thus does not support the conclusion of the authors. In addition, an important control is missing. How does the heat shock affect the morphology of AIAs in wt or ctbp-1 animals, without the hs-construct?*

    We agree with this comment and have updated the manuscript to clarify that suggestion of the activity of CTBP-1 in preventing further disruption of AIA morphology is speculative. We will conduct the suggested control experiment and include the results in a revised version of the manuscript.

    Apart from the above, all strong claims by the authors are valid. In addition, the authors suggest a mechanism, where CTBP-1 regulates the function of the EGL-13 transcription factor in AIA and that overexpression of CEH-28 in AIA contributes to the olfactory adaptation defect observed in the ctbp-1 mutant animals. These mechanistic speculations could be relatively easily strengthened by two additional experiments. One, does ctbp-1 loss of function affect egl-13 expression? The model presented in Fig 8 suggests that egl-13 expression levels are not affected, but from the data in the paper it is not even clear of egl-13 is expressed in AIA. Whether egl-13 is expressed in AIA, and if its expression levels are affected by mutation of ctbp-1 could be tested using egl-13::gfp expressing animals.

    This is an excellent suggestion and experiments we had been attempting already. We will include findings from these experiments once they are complete in a revised version of the manuscript.

    __ __Two, does overexpression of ceh-28 cause an olfactory adaptation defect? This could be tested by cell specific overexpression of ceh-28 in AIA.

    This is also a great suggestion. We will conduct this experiment and include the findings in a revised manuscript.

    *The data and the methods have been presented in such a way that they can be reproduced. I do have some doubts with regard to the statistical analysis. The authors report that statistical analysis involved unpaired t-tests. But as all results involve the analysis of data from 3-5 different strains, a multiple sample analysis should be used. To correct for the number of samples, one should first use an ANOVA to test for statistical differences, followed by a post hoc analysis to identify those that are significantly different.

    We agree with this criticism. We have replaced instances of multiple sample analyses with a one-way ANOVA test followed by Tukey’s multiple test correction. The current version of the manuscript reflects these changes in figures, figure legends and in the Materials and Methods.

    Reviewer #2:

    **Major comments:**

    1. The paper is well written and figures are clearly organized. The authors made suitable conclusions based on the data provided. Materials and methods are appropriately described for reproductivity.*

    *2. It would strengthen the model (Figure 8) by testing physical interaction between CTBP-1 and EGL-13 in AIA using BiFC.

    We agree and are currently attempting such experiments. Meaningful results from these experiments will be included in a revised manuscript.

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    **Major comments:**

    • The key conclusions of this manuscript are highly convincing and are supported by multiple mutant alleles and rescue experiments.*
    • There are certain claims in the manuscript that need to be clarified (detailed below).*
    • No additional experiments are essential to support the claims of the paper.*

    *- Most of the data and the methods presented well - however a Table listing genes identified in the AIA-specific RNA Seq is required. The GEO accession number has been made available for the RNA Sequencing data however listed the genes identified would aid the reader. Were ctbp-1 and egl-13 shown to be expressed in the AIAs using this approach?

    We have included such a table, replacing Fig. S6 (which previously showed only ceh-28 expression) with a table listing expression of all confirmed hits from the scRNA-Seq experiment. ctbp-1 and egl-13 were also found to be expressed in the AIA neurons in this scRNA-Seq experiment.

    *- No evidence is presented that EGL-13 is expressed in the AIAs?

    As noted above, the scRNA-Seq experiment showed *egl-13 *expression in the AIAs. We also will assay egl-13 expression in the AIAs using a GFP reporter and include the results in a revised manuscript.

    - Can the authors comment and include in the manuscript information regarding whether the promoters of AIA-expressed genes that are regulated by EGL-13 contain EGL-13 binding sites? Also, are the promoters of AIA-expressed genes not regulated by EGL-13 missing these sites?

    We have added such information to the manuscript. Briefly, our analysis identified no promising candidates for EGL-13 binding sites in the promoter regions of either ceh-28 or acbp-6, suggesting that regulation of these by EGL-13 is likely indirect. Further, no previous work has indicated that either of these genes is regulated directly by EGL-13, although in the case of acbp-6 little is known about this gene or the ways in which it is regulated. However, the claim that EGL-13 regulates expression of acbp-6 and ceh-28 indirectly is speculative and is not a conclusion of this current work.

    __ __- Experiments and statistical analysis are adequate.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    In this manuscript, Saul et al. found that the CTB-1 transcriptional co-repressor acts cell-autonomously to maintain aspects of AIA neuronal fate, morphology and function. They found that CTBP-1 utilizes the Sox transcription factor EGL-13 to transcriptionally repress specific genes in the AIA neurons. This work proposes that CTBP-1 and other co-repressors play critical roles in selectively maintaining or repressing expression of specific genes.

    Major comments:

    • The key conclusions of this manuscript are highly convincing and are supported by multiple mutant alleles and rescue experiments.
    • There are certain claims in the manuscript that need to be clarified (detailed below).
    • No additional experiments are essential to support the claims of the paper.
    • Most of the data and the methods presented well - however a Table listing genes identified in the AIA-specific RNA Seq is required. The GEO accession number has been made available for the RNA Sequencing data however listed the genes identified would aid the reader. Were ctbp-1 and egl-13 shown to be expressed in the AIAss using this approach?
    • No evidence is presented that EGL-13 is expressed in the AIAs?
    • Can the authors comment and include in the manuscript information regarding whether the promoters of AIA-expressed genes that are regulated by EGL-13 contain EGL-13 binding sites? Also, are the promoters of AIA-expressed genes not regulated by EGL-13 missing these sites?
    • Experiments and statistical analysis are adequate.

    Minor comments:

    I list below a number of changes and typographical errors that will improve the manuscript.

    Page 11 Line 235 - the authors state that ctbp-1 L4s have an increased attraction to butanone. As the chemotaxis index is 0 for the ctbp1- mutant compared to -0.5 in WT I understand what the authors mean hear but the statement of "increased attraction" suggests that ctbp1- mutants are attracted to butanone when they are actually ambivalent to it.

    Page 12 Line 248 - change functioning to functional

    Page 18 Line 397 - it would be helpful to the reader if the authors referred back to the ctbp1- mutant data (Figure 5) for comparison in Fig 7D.

    Page 19 Line 404 - remove the word causally

    Page 19 Line 414 "However, while conditioned ctbp-1 ceh-28 double mutants appeared similar to both the wild type and ctbp-1 single mutants at the L1 stage (Fig. 7I-J), these double mutants displayed an intermediate phenotype between wild-type and ctbp-1 animals for adaptation at the L4 larval stage (Fig. 7K-L).

    This sentence is confusing as the ctbp-1 ceh-28 phenotype is not significant different to the ctbp-1 single mutant.

    Page 50 Line 1001 - change mlg-1 to mgl-1

    Figure 7A-C - please label with the genotype examined.

    Significance

    • This work identifies a function for the transcriptional corepressor CTBP-1 in controlling the expression of a subset of genes in the AIA neurons. It suggests that CTBP-1 may play a similar role in controlling subsets of gene expression in diverse neuronal classes. This would be interesting to examine in single cell sequencing experiments of all C. elegans neurons.
    • This work adds to the literature that describes CTBP-1 functions in the C. elegans nervous system. It also speculates that other transcriptional co-repressors play similar functions in other cells and tissues in other organisms.
    • An audience with interests in cell fate determination and the function of specific gene regulatory modules that control subsets of genes within a cell.
    • My field of expertise is C. elegans neurobiology (axon guidance and cell fate) and I am therefore well-qualified to review this manuscript.

    Referee Cross-commenting

    Comments from other reviewers are fair. I am happy with the overall conclusions.

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    Referee #2

    Evidence, reproducibility and clarity

    In this paper, the authors identified several mutations from a forward genetic screen in the transcriptional corepressor gene ctbp-1 that cause mixexpression of a M4 neuronal marker in the two AIA interneurons in C. elegans. ctbp-1 mutant AIA neurons also display a defect in morphology and sensory function. The penetrance and severity of these defects in gene expression, morphology, and function progressively increase with age. Their data suggests that ctbp-1 acts cell-autonomously and in older worms to maintain gene expression, morphology, and function in AIA neurons. Single-cell RNA sequencing was performed to identify changes in AIA transcriptional profiles between wild type and ctbp-1 mutants. Using the data from AIA transcriptional profiles, they showed that ctbp-1 mutant AIA neurons lose the expression of two genes characteristic of the adult AIA while misexpress at least two genes uncharacteristic of AIA. Taken together, their findings demonstrate that ctbp-1 acts to maintain the AIA identity at the level of gene expression, morphology, and function, while ctbp-1 does not act to establish the AIA cell identity. Furthermore, the authors identified a few mutations of a SOX family transcription factor gene egl-13 from a froward genetic screen that suppress the ctbp-1 mutant phenotype. The authors conclude their results that ctbp-1 maintains AIA function and some aspects of AIA gene expression by antagonizing egl-13 function and that ctbp-1 maintains AIA morphology through pathways independently of egl-13.

    Major comments:

    1. The paper is well written and figures are clearly organized. The authors made suitable conclusions based on the data provided. Materials and methods are appropriately described for reproductivity.
    2. It would strengthen the model (Figure 8) by testing physical interaction between CTBP-1 and EGL-13 in AIA using BiFC.

    Minor comments:

    1. The authors mentioned a previous finding that the mammalian ortholog of EGL-13, SOX6, interacts with the mammalian ortholog of CTBP-1, CtBP2. The authors should also discuss the function of interacting SOX6 and CTBP-1 in mammalian systems.
    2. It would be good to increase the font size of some figures and tables for easier reading.

    Significance

    This study identifies roles of conserved transcriptional corepressor CTBP-1 and a SOX family transcription factor gene egl-13 from unbiased forward genetic screens in the maintenance of AIA interneurons in C. elegans.

    Since CTBP-1 and EGL-13 have mammalian orthologs, although the roles of their mammalian orthologs were not discussed, this study may have broad implications for development in a range of organisms.

    The findings of this study will be of interest to a broad audience in the field of developmental biology, particularly in transcriptional regulation of cell identity maintenance.

    I have expertise in transcriptional regulation of sensory neuron diversification using C. elegans as a model. I am comfortable about evaluating this manuscript.

    Referee Cross-commenting

    I agree with the comments from reviewers 1 and 3.

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    Referee #1

    Evidence, reproducibility and clarity

    In this manuscript, Saul et al identify the transcriptional corepressor ctbp-1 as a regulator of ceh-28::gfp expression in the AIA neurons of the nematode C. elegans. They find 18 independent mutants in this gene, including several presumptive null alleles. Using cell specific rescue and temporal expression of the ctbp-1 gene, the authors show that ctbp-1 acts cell autonomously in AIA to regulate ceh-28 expression, and can do so in young adult animals. Next, using various reporters they show that the AIAs do not transdifferentiate to M4-like cells, but the AIAs do show morphological defects, which increase with age of the animal. Using behavioral experiments the authors next determine the functionality of the AIAs in the ctbp-1 mutant animals. They find that loss of ctbp-1 in AIA affects the function of the AIAs and that ctbp-1 does so on young adult animals. The authors conclude the characterization of the AIAs of ctbp-1 mutant animals by identifying several other genes whose expression is misregulated in ctbp-1 animals, using a single cell RNAseq experiment, confirmed using gfp-fusion constructs. These experiments identity one other gene, acbp-6, that is misexpressed in the AIAs of L4 ctbp-1 animals and 2 genes, sra-11 and glr-2 that are normally expressed in AIA, but not in ctbp-1 animals.

    To find out how ctbp-1 regulates gene expression in AIA, the authors perform a genetic suppressor screen and show that loss of function of egl-13 suppresses the ceh-28::gfp misexpression in AIA in ctbp-1 mutants. They show that egl-13 functions cell-autonomously in the AIAs. They find it does not suppress the morphological defects of the AIAs in ctbp-1 mutants, but it does suppress the effect of ctbp-1 loss of function on olfactory adaptation. In addition, mutation of egl-13 suppressed the misexpression of acbp-6, but not that of sra-11 and glr-2. Finally, the authors show that the olfactory adaptation defect observed in ctbp-1 mutant animals can be partially suppressed by inactivating ceh-28 suggesting that the behavioral defect is caused in part by overexpression of ceh-28.

    The manuscript is very well written and results have been very clearly presented. The key conclusions drawn by the authors are convincing. However, one of the claims by the authors is not supported by the data. In lines 206-215 the authors discuss experiments where they visualized the morphology of the AIAs in ctbp-1 mutants where ctbp-1 expression is restored temporally in the L4-young adult stage using a heat-shock promoter construct. The authors conclude that "ctbp-1 can act ... in older worms to maintain aspects of AIA morphology in a manner similar to AIA gene expression." However, the data presented in Fig. 3I-L show no statistically significant difference between ctbp-1 mutants and mutants with the HS-construct, either with and without heat shock. Thus, although there seems to be some effect of the heat shock, this is not significant and thus does not support the conclusion of the authors. In addition, an important control is missing. How does the heat shock affect the morphology of AIAs in wt or ctbp-1 animals, without the hs-construct?

    Apart from the above, all strong claims by the authors are valid. In addition, the authors suggest a mechanism, where CTBP-1 regulates the function of the EGL-13 transcription factor in AIA and that overexpression of CEH-28 in AIA contributes to the olfactory adaptation defect observed in the ctbp-1 mutant animals. These mechanistic speculations could be relatively easily strengthened by two additional experiments. One, does ctbp-1 loss of function affect egl-13 expression? The model presented in Fig 8 suggests that egl-13 expression levels are not affected, but from the data in the paper it is not even clear of egl-13 is expressed in AIA. Whether egl-13 is expressed in AIA, and if its expression levels are affected by mutation of ctbp-1 could be tested using egl-13::gfp expressing animals.

    Two, does overexpression of ceh-28 cause an olfactory adaptation defect? This could be tested by cell specific overexpression of ceh-28 in AIA.

    These are relatively simple experiments that would not take much time or investments, but would strengthen or clarify the model presented.

    The data and the methods have been presented in such a way that they can be reproduced. I do have some doubts with regard to the statistical analysis. The authors report that statistical analysis involved unpaired t-tests. But as all results involve the analysis of data from 3-5 different strains, a multiple sample analysis should be used. To correct for the number of samples, one should first use an ANOVA to test for statistical differences, followed by a post hoc analysis to identify those that are significantly different.

    Minor comments:

    Page 7, in the heat shock rescue experiment that authors conclude that ctbp-1 acts "in older worms" to prevent expression of ceh-28 in AIA. "Older" is quite unspecific. Please be specific, i.e. in L4-young adult animals. The same applies to various other phrases where "older" worms are mentioned. Line 229, the authors state that animals were "briefly starved". Please be precise and indicate how long the animals were starved.

    Significance

    Most studies that address cell fate, focus on the first phase where cell fate is determined. How cell fate is maintained is far less well understood. This manuscript convincingly identifies two transcription regulators that are important for cell fate maintenance, both a transcriptional repressor and an activator. The manuscript provides first clues as to how this process functions, and as such provides important conceptual insights. These not only apply to the worm, C. elegans, but as these are strongly conserved proteins, probably also provide a firm basis for our understanding of cell fate maintenance mechanisms in higher organisms including mammals. In addition, this study reports an excellent model that can be used to further unravel this mechanism. As such, I expect that this manuscript will be of interest to a broad range of scientists, interested in cell fate determination and maintenance and transcriptional control.

    My expertise lies in C. elegans behavior, where we focus on identification of the molecular and cellular mechanisms that allow C. elegans to respond to its environment even in changing circumstances. In addition, we study the mechanisms of cell fate determination and maintenance in C. elegans sensory neurons.

    Referee Cross-commenting

    I agree with the comments of reviewers 2 and 3.

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