A transcription factor toggle switch determines differentiated epidermal cell identities in Hydra

  1. Jaroslav Ferenc
  2. Marylène Bonvin
  3. Panagiotis Papasaikas
  4. Jacqueline Ferralli
  5. Clara Nuninger
  6. Charisios D. Tsiairis

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Abstract

In Hydra , a simple cnidarian model, epithelio-muscular cells play a crucial role in shaping and maintaining the body architecture. These cells are continuously renewed as undifferentiated cells from the body’s mid-region get displaced toward the extremities, replacing shed, differentiated cells and adopting specific identities. This ongoing differentiation, coupled with the maintenance of distinct anatomical regions, provides an ideal system to explore the relationship between cell type specification and axial patterning. However, the molecular mechanisms governing epithelial cell identity in Hydra remain largely unknown. In this study, we describe a double-negative feedback loop between the transcription factors Zic4 and Gata3 that functions as a toggle switch to control epidermal cell fate. Zic4 is activated by Wnt signaling from the mouth organizer and triggers battery cell specification in tentacles. In contrast, Gata3 promotes basal disk cell identity at the aboral end. Functional analyses demonstrate that Zic4 and Gata3 are mutually antagonistic; suppression of one leads to the dominance of the other, and vice versa , resulting in ectopic cell specification. Notably, simultaneous knockdown of both factors rescues the phenotype, indicating that it is the balance between these transcription factors, rather than their absolute levels, that dictates cell identity. This study highlights the mechanisms by which distinct cellular identities are established at Hydra body termini and reveals how cell fate decisions are coordinated with axial patterning.

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    Reply to the reviewers

    Manuscript number: RC-2024-02831

    Corresponding author(s): Charisios Tsiairis

    1. General Statements [optional]

    We are very pleased that all three reviewers found our work to be solid, well-supported by the data, and free of major flaws. It is particularly gratifying that they did not request additional experimental work to support our conclusions. Instead, their comments focused on clarifications, textual improvements, and refinements in data presentation, which we have carefully addressed.

    We have made revisions to improve the clarity of the manuscript, incorporating insightful suggestions from the reviewers. These include refining key explanations, adjusting figure annotations, and modifying the structure of certain sentences. Additionally, we have addressed specific points regarding statistical significance, genome assembly references, and phylogenetic comparisons, ensuring that all aspects of our study are as precise and informative as possible.

    We are confident that these revisions have strengthened the manuscript.

    2. Point-by-point description of the revisions

    *Reviewer #1 (Evidence, reproducibility and clarity (Required)): *

    *Overall, the paper is well-written, the figures are easy to interpret, and the conclusions are well supported by the data. Most of the points discussed below could be addressed with simple text changes. *

    *General Points: *

    • The upregulation of Gata3 in response to Zic4 RNAi is relatively modest compared to the more pronounced upregulation of Zic4 following Gata3 knockdown, but this point is not really addressed. While these issues could be simply technical, they might also hint at additional layers of regulation that are not yet fully understood. *

    The observed differences in upregulation are primarily technical. Expression levels are measured relative to unperturbed tissue, and in the control, Zic4 expression in the foot is detected only at noise levels (see figure 2C). As a result, any increase in Zic4 expression upon Gata3 knockdown appears relatively high when normalized to the minimal control levels. In contrast, Gata3 is already present at detectable levels in control samples from the upper body, head, and tentacles (See Fig 2D). Therefore, while its upregulation following Zic4 RNAi appears more modest, we interpret this as a qualitative indication of increased gene expression in the absence of the opposing transcription factor. That said, we acknowledge the possibility of additional regulatory layers contributing to these differences.

    • Extending the time course would strengthen the conclusion that, in the Gata3 knockdown, the existing basal disk cells remain stable while body column cells migrating into the region differentiate into tentacle cells. If this hypothesis is correct, one would predict that by approximately 20 days, the basal disk cells would be completely replaced. *

    This is a valid point; however, the interpretation is complicated by the technical limitations of RNAi-based knockdown rather than a complete knockout of Gata3. Over time, the effect of RNAi diminishes, and we have observed that GFP expression returns within four weeks following GFP RNAi, indicating a temporal limit to RNAi-mediated knockdown. Therefore, while an extended time course would be informative, the transient nature of the knockdown makes it challenging to definitively track long-term cell replacement dynamics.

    • The conclusion that tentacle cells transdifferentiate into basal disc cells in the Zic4 knockdown may require more nuance, as only the tips of the tentacles express peroxidase. Do the more proximal regions of the tentacle express peduncle markers? *

    We appreciate the reviewer’s comment. In our previous publication (Vogg et al., 2022), we provided evidence supporting this phenomenon. As demonstrated in our data published there, markers of the peduncle, rather than the basal disc—such as manacle (gene ID 100212761) (Bridge et al., 2000) and Bmp5-8 (gene ID 100206618) (Reinhardt et al., 2004)—are also upregulated, suggesting a transition towards a peduncle-like state. However, we opted not to elaborate on this aspect in the current manuscript to maintain focus and avoid redundancy with previously published findings.

    *Specific Points: *

    *Figure 1A, Figure 4E: The pictorial representation of Zic4 expression may need to be revised, as in situ hybridization data from Vogg et al., 2022, suggests that Zic4 is absent from the hypostome and tentacle tips. While in situ hybridization can sometimes lack precision due to variability in staining protocols and subjective decisions on when to stop the reaction, this observation aligns with scRNA-seq data, which also indicates a lack of Zic4 expression in the hypostome and tips of the tentacles. *

    Our intention was to illustrate the general presence of Zic4 in the oral domain, but we acknowledge the reviewer’s point that this could be misleading regarding its precise expression pattern. To address this concern, we have updated the figure panels to more accurately reflect the available in situ hybridization and scRNA-seq data.

    *Figure 1 Legend: For panel D, the legend says "data taken from 28" but the references are not numbered. Same problem for panel E legend. *

    We thank the reviewer for catching this error. We have now corrected the references, replacing the numbering with the first authors' last names and publication dates.

    Figure 1D: There may be a mistake in the Hydra body part labeling. Is "B" supposed to be "P" for peduncle?

    We appreciate the reviewer’s observation. The label refers to the budding zone, and we acknowledge our omission in specifying this. We have now updated the figure and its legend to clarify this.

    *Figure 1 Panel E: Please provide clarification regarding what each box means. Are these 8 replicates of the same condition, or are these the proximal and distal regions of the tentacles as was collected in the Vogg paper? *

    We appreciate the reviewer’s request for clarification. These conditions are indeed similar to those in the previously published Vogg et al. paper. The boxes in the figure represent proximal and distal tentacle regions, each with four replicates. We have now updated the figure and its legend to make this explicit.

    *Figure 2A: Consider using the co-expression stats from Fig S2, which are very informative. *

    *We added the percentage of cells expressing Zic4, Gata3 and both genes on the panel. *

    *Figure 2E, F: It would be more intuitive to group each experimental sample with its corresponding control. *

    To make the figure clearer, we modified it and grouped each experimental sample with its corresponding control.

    *Figure 2C-F: Consider conducting statistical tests of significance between control and treatment groups. *

    We have now expanded the statistical analyses, ensuring that significance tests are presented in all relevant instances. However, we note that while statistical significance is important, it should be interpreted alongside other factors such as the magnitude of the effect, consistent trends across replicates, and biological relevance. Additionally, high standard deviations in certain conditions may influence absolute p-values, and we encourage consideration of the broader context of the data when interpreting these results.

    *Figure 2 E - Considering the error bars, Gata3 upregulation in response to Zic4 knockdown does not look significant based on qPCR. Showing the significance of the up-regulation in the RNA-seq data may be more convincing. (I believe RNA-seq to be more reliable anyway). *

    We understand the reviewer’s concern. The p-value for the qPCR data is slightly above 0.05, primarily due to high standard deviation. As the reviewer notes, qPCR on RNAi samples can be noisy, so the data should be interpreted in context. Importantly, the consistent qualitative increase in Gata3 levels after Zic4 knockdown aligns with the RNA-seq results, which, as the reviewer correctly points out, provide a more reliable measurement. Additionally, qPCR samples include a broader portion of head tissue, likely diluting the Gata3 signal from the tentacles and contributing to the observed variability.

    *Figure S2: Might be helpful to show co-expression UMAPs here, like what is shown in Figure 2A. *

    We appreciate the reviewer’s suggestion. However, we believe that displaying co-expression UMAPs for Zic4 would be redundant. Additionally, for genes with greater positional overlap, such as FoxI1 and Nfat5, co-expression UMAPs make visualization more challenging. To ensure clarity and optimize the interpretability of the data, we have chosen to present the expression profiles of each gene separately.

    *Page 4: "Interestingly, a similar binary choice pattern appears in certain neuronal lineages as well. A recent study demonstrated the involvement of Gata3 in specifying neurons at the aboral end (Primack et al. 2023), suggesting that this cross-regulation between Zic4 and Gata3 may extend beyond the epidermal lineage." Just a note that this paper shows expression, but doesn't show function as the statement implies, so the statement should be changed accordingly. *

    Indeed, the study does not focus on the functional role of Gata3 in these neurons. We have revised the sentence, replacing "involvement of Gata3 in specifying neurons" with "expression expression of Gata3 in neurons emerging*" to more accurately reflect the study’s findings. *

    *Page 10: "Transcription Factor Binding site analysis... Hydra promoter sequences were compiled from the NCBI Hydra RP 105 assembly." Authors should provide a repository identifier for the genome they are using. Based on the information provided, it appears the authors are using Genome assembly "Hydra_RP_1.0" RefSeq GCF_000004095.1. However, that genome assembly has been suppressed for the following reason: "superseded by newer assembly for species". Authors should consider updating the reference assembly they are using to map their sequencing data and identify promoter sequences. *

    We appreciate the reviewer’s concern. However, we have chosen to use the Hydra_RP_1.0 assembly for Figure 1 to maintain consistency with previously published data, which were also mapped to this assembly. Since these publications predate the newer assembly, using the same reference ensures comparability in our analysis. Importantly the assembly used is still downloadable and accessible to every researcher. That said, for the phylogenetic analysis in Figure 2, we have used the latest available genome assemblies and annotations for all species, including Hydra. We have now clarified this in the Methods section.

    *The paper makes great use of the Hydra scRNA-seq data set! Minor point, when referring to the Hydra scRNA-seq data set, please cite Siebert et al., 2019 (data collection) and Cazet et al., 2023 (analysis that is being used in this paper). *

    We appreciate the reviewer’s suggestion and have updated the references accordingly to include Siebert et al., 2019, for data collection and Cazet et al., 2023, for the analysis used in this paper.

    Something to keep in mind: To an audience without expertise in Hydra cell type morphology, the nematocyte marker HCR will likely be more convincing than the actin staining in Figure 3D to identify and quantify nematocytes.

    We agree with the reviewer that the nematocyte marker HCR provides a more specific identification of nematocytes. This is why we have also used the nematocilin marker in separate samples. However, actin staining adds important information on the morphology of the surrounding epithelial cells, which become indistinguishable from battery cells in Gata3 KDs. Unfortunately, combining actin staining with HCR is technically challenging, as the tissue preparation protocols for these two approaches are not compatible, and we have therefore decided to show both stainings next to each other.

    *Minor Wording Issues: *

    *Page 2. "However, the mechanism by which Zic4 prevents the battery cell program from misexpression in normal tentacles remained unclear." Could read more clearly as: However, the mechanism by which Zic4 prevents the misexpression of the battery cell program in normal tentacles remained unclear. *

    We have made the suggested change.

    *Page 2. "Potential candidates for this function could be found among TFs with highly enriched binding sites in the dataset, which are themselves Zic4 targets." Could read more clearly as: We reasoned that this intermediary factor, likely a target of Zic4, would be a transcription factor with highly enriched binding sites in the dataset. *

    We are grateful for the suggestion, we have changed the text accordingly.

    *p3-4. "Q-PCR performed on dissected oral and aboral body regions confirmed this finding (Fig. 2C-D)" It is unclear which "finding" is being confirmed. *

    We are referring to the upregulation of gata3 expression in tentacles upon Zic4 knockdown. To make this clearer, we have revised the wording to: “Q-PCR performed on dissected oral and aboral body regions confirmed __the upregulation of gata3 upon Zic4 knockdown __(Fig. 2C-D).”

    *Reviewer #1 (Significance (Required)): *

    *This compelling study from the Tsiairis lab uncovers a double-negative feedback loop between the transcription factors Zic4 and Gata3, functioning as a toggle switch to control oral and aboral fates in Hydra's epidermal lineage. Addressing fundamental questions in developmental biology, this research sheds light on the mechanisms underlying cell fate determination in relationship to their spatial organization. In Hydra, Wnt signaling, a conserved pathway critical for establishing primary body axes, promotes oral fate, emanating from an organizer at the oral end. Hydra body column epidermal cells can differentiate into distinct cell types, including oral battery cells and aboral basal disk cells, but the regulatory mechanisms remained elusive. Recent research from the Tsiairis lab identified Zic4 as a direct Wnt signaling target necessary for repressing basal disk-specific genes. Knocking down Zic4 caused battery cells to transform into basal disk cells, though Zic4 did not directly activate basal disk-specific genes, pointing to an intermediary regulator. This study identifies Gata3 as a key regulator of basal disk gene expression, as it is highly expressed at the aboral end, is inversely correlated with Zic4, and is upregulated in Zic4 knockouts. Functional experiments revealed mutual inhibition between Zic4 and Gata3: knocking down Gata3 led to differentiation of battery cells at the aboral end, while simultaneous knockdowns of Zic4 and Gata3 rescued the phenotypes of individual knockdowns. These findings demonstrate a finely tuned balance between Zic4 and Gata3 in regulating cell fate along the oral-aboral axis in Hydra. This paper therefore offers new insights into the spatial organization of cell type specification in Hydra and into broader principles of cell fate determination. *

    *We appreciate the reviewer’s thoughtful summary and recognition of our study’s significance. *

    *Reviewer #2 (Evidence, reproducibility and clarity (Required)): *

    *Summary: *

    *The authors use the freshwater hydrozoan Hydra as a model to investigate mechanisms of cell fate decisions in the context of terminal epithelial differentiation. The epithelia migrates towards the extremities of the animal and takes on one of two fates: elongated battery cells that house the cnidocytes ( stinging cells ) in the oral ( head ) end of the animal, or more compact secretory basal disc cells at the aboral ( foot ) end. In this manuscript the authors build on previous work that showed the transcription factor Zic4 is necessary for battery cell formation. The authors use in situ hybridization and additional labelling techniques to assess cell fate under a variety of conditions. The authors first screen for Zic4 binding sites in the promoter regions of aboral genes that previously were demonstrated to be up-regulated in response to Zic4 knockdown, and survey publicly available expression databases to identify GATA3 as a candidate transcription factor that shows complementary expression patterns. The authors also screen the promoter regions of Zic4 and GATA3 from a number of other cnidarians and find reciprocal binding sites in all but one case. This is interpreted by the authors as evidence for a Zic4/GATA3 cnidarian regulatory motif. The authors demonstrate that KD of GATA3 results in the opposite phenotype: ectopic differentiation of oral battery cells, and that animals with perturbed GATA3 function fail to regenerate the aboral basal disk cells but rather show oral battery cell phenotype. Further, KD of both genes (Zic4: battery cells and GATA3: pedal disc cells) results in a rescue of the phenotype of either single KD, thereby illustrating that together these two genes function as a negative feedback loop controlling the terminal differentiation of the ectodermal epithelia. *

    *Major comments: *

    *- Are the key conclusions convincing? *

    *The key conclusions are convincing. *

    *- Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether? *

    *The cross species comparison of binding sites is insightful, but is presented very early in the manuscript. This would be better placed as a final piece, to place the Hydra-specific findings in a larger context. *

    *- Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation. *

    *No. *

    *- Are the data and the methods presented in such a way that they can be reproduced? *

    *Yes, *

    *- Are the experiments adequately replicated and statistical analysis adequate? *

    *Yes. *

    *Minor comments: *

    *- Specific experimental issues that are easily addressable. *

    *None. *

    *- Are prior studies referenced appropriately? *

    *Yes. *

    *- Are the text and figures clear and accurate? *

    *Yes. The figures are very nice. *

    *- Do you have suggestions that would help the authors improve the presentation of their data and conclusions? *

    *1) Move the phylogenetic comparisons to the end *

    *2) Similarly, in the section on GATA3 KD, present the normal condition first, and then the regeneration experiment results. *

    We thank the reviewer for their positive assessment and constructive suggestions. Below, we comment on each point:

    • Placement of cross-species comparison:* This suggestion concerns the emphasis and structure of the manuscript. We appreciate the reviewer's interest in the evolutionary aspects of our work. However, we believe that moving this analysis to the end would dilute the main message, which is reinforced by the schematic in Figure 4E-F. We aim to conclude with the experimental results demonstrating the minimization of phenotypic consequences when both factors are knocked down. Therefore, we have chosen to retain the cross-species comparison in its current position to emphasize the conservation of the double-negative interaction before presenting the functional consequences of its perturbation.*
    • Reordering of Gata3 KD results:* We understand the rationale behind this suggestion. However, our sequencing is guided by the fact that foot regeneration deficiency under Gata3 kd has already been documented and presented in previous work (Ferenc et al., 2021). For this reason, we begin with that reference, then build upon it with a deeper examination of the phenotype.*

    We are grateful for the reviewer’s feedback and for recognizing the clarity of our figures and analysis.

    ***Referee cross-commenting** *

    *I have read the other two reviews and find that we are all in agreement that the work presented in this manuscript is sound and is a valuable scientific contribution. I would encourage the authors to consider my own suggests for order of presentation of data, to retain a specific to broad theme (normal then regeneration / hydra then comparisons) and to incorporated the detailed corrections highlighted by reviewer 1. *

    *Regarding reviewer 3's comment regarding SoxA in cnidarians. This is likely true and the nomenclature of the gene likely comes from an automated pipeline to infer gene identities. Unless the authors follow up on this gene, I don't think the onus is on the authors to confirm the identity. *

    We appreciate Reviewer’s #3 remark about the nuance of transcription factor homology. The situation is exactly as described here by Reviewer #2 - The gene names in Figure 1 are based on the results of NCBI automated homology annotation, which we have now clarified in a note in the legend of Figure 1.

    *Reviewer #2 (Significance (Required)): *

    *- Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field. *

    *This paper is a beautiful illustration of the importance of relative gene expression levels in controlling cell fate decisions. Together with their previous works, the role of both transcription factors in specifying one of two possible terminal fates is very clearly illustrated. The final observation, that a mutual knockdown of both factors leads to a rescue of the polarity of the cell type balance is an excellent example of the importance of relative gene expression levels in controlling homeostatic balance between two mutually exclusive cell fates. *

    *- Place the work in the context of the existing literature (provide references, where appropriate). *

    *The manuscript does a good job of placing the work into the appropriate context. *

    *- State what audience might be interested in and influenced by the reported findings. *

    *Readers with interest in gene regulation, cell specification, and mechanisms of cell type diversification would find these results of interest. *

    *- Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate. *

    *Comparative invertebrate embryogenesis; Single cell transcriptomics; Cell and tissue evolution *

    We greatly appreciate the reviewer’s positive feedback and recognition of our study's focus on gene expression in cell fate decisions. We're pleased that our findings on the mutual knockdown and the broader context were well received. Thank you for highlighting the relevance of our work to gene regulation and cell specification.

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

    *Ferenc et al. have studied the role of transcription factors Zic4 and Gata3 in Hydra epithelial cell fate decision. The Tsiairis team has published a paper recently in which they had studied the role of Zic4 in promoting tentacle formation. Here, they discover a negative feedback loop between Zic4 and Gata3 in the context of epithelial cell differentiation. The authors used computational techniques to identify Zic4 binding sited in Hydra promoters of genes that are upregulated in basal disks, known from a previous study, and identified eight candidate genes. Previous studies were also used to narrow down potential Zic4 targets. They argue that Gata3 appears as a strong candidate to be suppressed by Zic4 in the head and being expressed in the foot. Knockdown experiments, followed by qPCR revealed that Gata3 and Zic4 expression is mutually exclusive such that the one represses the other. Next, they report that Gata3 RNAi results in ectopic battery cells at the lower body column, although basal disk cells maintained their identity following Gata3 knockdown. Finally, knocking down both Gata3 and Zic4 resulted in a more normal phenotype, as predicted if a negative feedback loop existed between the two. *

    *A minor comment: one of the predicted Zic4 targets is a gene called Sry. Sry is a mammalian male determinant and a SOX-related protein (SoxA). I was wondering if the authors performed phylogenetic analysis or simply took a BLAST hit as the source for this gene's name. I am unaware of SoxA-like genes in cnidarians . Therefore, I would recommend performing a SOX phylogeny and renaming it according to its closest relatives, which probably won't be Sry. *

    The naming of the gene as Sry was indeed based on the NCBI automated homology annotation, and we have clarified this in the revised manuscript. Since we did not pursue further analysis of this gene, we believe that a deeper phylogenetic analysis may not be necessary and could potentially divert attention from the main focus of our study on Gata3's role.

    *Reviewer #3 (Significance (Required)): *

    *This work closes some gaps that remained after publication of previous research by the Tsiairis lab and others. The data are of high quality, solid, and support the authors' conclusions. The manuscript is of general interest for developmental biologists and evodevo workers. *

    We thank the reviewer for the thoughtful assessment of our work. We appreciate their feedback and the recognition of the quality and significance of our findings.

  2. Review Commons

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    Referee #3

    Evidence, reproducibility and clarity

    Ferenc et al. have studied the role of transcription factors Zic4 and Gata3 in Hydra epithelial cell fate decision. The Tsiairis team has published a paper recently in which they had studied the role of Zic4 in promoting tentacle formation. Here, they discover a negative feedback loop between Zic4 and Gata3 in the context of epithelial cell differentiation. The authors used computational techniques to identify Zic4 binding sited in Hydra promoters of genes that are upregulated in basal disks, known from a previous study, and identified eight candidate genes. Previous studies were also used to narrow down potential Zic4 targets. They argue that Gata3 appears as a strong candidate to be suppressed by Zic4 in the head and being expressed in the foot. Knockdown experiments, followed by qPCR revealed that Gata3 and Zic4 expression is mutually exclusive such that the one represses the other. Next, they report that Gata3 RNAi results in ectopic battery cells at the lower body column, although basal disk cells maintained their identity following Gata3 knockdown. Finally, knocking down both Gata3 and Zic4 resulted in a more normal phenotype, as predicted if a negative feedback loop existed between the two.

    A minor comment: one of the predicted Zic4 targets is a gene called Sry. Sry is a mammalian male determinant and a SOX-related protein (SoxA). I was wondering if the authors performed phylogenetic analysis or simply took a BLAST hit as the source for this gene's name. I am unaware of SoxA-like genes in cnidarians . Therefore, I would recommend performing a SOX phylogeny and renaming it according to its closest relatives, which probably won't be Sry.

    Significance

    This work closes some gaps that remained after publication of previous research by the Tsiairis lab and others. The data are of high quality, solid, and support the authors' conclusions. The manuscript is of general interest for developmental biologists and evodevo workers.

  3. Review Commons

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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    The authors use the freshwater hydrozoan Hydra as a model to investigate mechanisms of cell fate decisions in the context of terminal epithelial differentiation. The epithelia migrates towards the extremities of the animal and takes on one of two fates: elongated battery cells that house the cnidocytes ( stinging cells ) in the oral ( head ) end of the animal, or more compact secretory basal disc cells at the aboral ( foot ) end. In this manuscript the authors build on previous work that showed the transcription factor Zic4 is necessary for battery cell formation. The authors use in situ hybridization and additional labelling techniques to assess cell fate under a variety of conditions. The authors first screen for Zic4 binding sites in the promoter regions of aboral genes that previously were demonstrated to be up-regulated in response to Zic4 knockdown, and survey publicly available expression databases to identify GATA3 as a candidate transcription factor that shows complementary expression patterns. The authors also screen the promoter regions of Zic4 and GATA3 from a number of other cnidarians and find reciprocal binding sites in all but one case. This is interpreted by the authors as evidence for a Zic4/GATA3 cnidarian regulatory motif. The authors demonstrate that KD of GATA3 results in the opposite phenotype: ectopic differentiation of oral battery cells, and that animals with perturbed GATA3 function fail to regenerate the aboral basal disk cells but rather show oral battery cell phenotype. Further, KD of both genes (Zic4: battery cells and GATA3: pedal disc cells) results in a rescue of the phenotype of either single KD, thereby illustrating that together these two genes function as a negative feedback loop controlling the terminal differentiation of the ectodermal epithelia.

    Major comments:

    • Are the key conclusions convincing?

    The key conclusions are convincing.

    • Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

    The cross species comparison of binding sites is insightful, but is presented very early in the manuscript. This would be better placed as a final piece, to place the Hydra-specific findings in a larger context.

    • Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.

    No.

    • Are the data and the methods presented in such a way that they can be reproduced?

    Yes,

    • Are the experiments adequately replicated and statistical analysis adequate?

    Yes.

    Minor comments:

    • Specific experimental issues that are easily addressable.

    None.

    • Are prior studies referenced appropriately?

    Yes.

    • Are the text and figures clear and accurate?

    Yes. The figures are very nice.

    • Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

    1. Move the phylogenetic comparisons to the end

    2. Similarly, in the section on GATA3 KD, present the normal condition first, and then the regeneration experiment results.

    Referee cross-commenting

    I have read the other two reviews and find that we are all in agreement that the work presented in this manuscript is sound and is a valuable scientific contribution. I would encourage the authors to consider my own suggests for order of presentation of data, to retain a specific to broad theme (normal then regeneration / hydra then comparisons) and to incorporated the detailed corrections highlighted by reviewer 1.

    Regarding reviewer 3's comment regarding SoxA in cnidarians. This is likely true and the nomenclature of the gene likely comes from an automated pipeline to infer gene identities. Unless the authors follow up on this gene, I don't think the onus is on the authors to confirm the identity.

    Significance

    • Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.

    This paper is a beautiful illustration of the importance of relative gene expression levels in controlling cell fate decisions. Together with their previous works, the role of both transcription factors in specifying one of two possible terminal fates is very clearly illustrated. The final observation, that a mutual knockdown of both factors leads to a rescue of the polarity of the cell type balance is an excellent example of the importance of relative gene expression levels in controlling homeostatic balance between two mutually exclusive cell fates.

    • Place the work in the context of the existing literature (provide references, where appropriate).

    The manuscript does a good job of placing the work into the appropriate context.

    • State what audience might be interested in and influenced by the reported findings.

    Readers with interest in gene regulation, cell specification, and mechanisms of cell type diversification would find these results of interest.

    • Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

    Comparative invertebrate embryogenesis; Single cell transcriptomics; Cell and tissue evolution

  4. Review Commons

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    Referee #1

    Evidence, reproducibility and clarity

    Overall, the paper is well-written, the figures are easy to interpret, and the conclusions are well supported by the data. Most of the points discussed below could be addressed with simple text changes.

    General Points:

    1. The upregulation of Gata3 in response to Zic4 RNAi is relatively modest compared to the more pronounced upregulation of Zic4 following Gata3 knockdown, but this point is not really addressed. While these issues could be simply technical, they might also hint at additional layers of regulation that are not yet fully understood.
    2. Extending the time course would strengthen the conclusion that, in the Gata3 knockdown, the existing basal disk cells remain stable while body column cells migrating into the region differentiate into tentacle cells. If this hypothesis is correct, one would predict that by approximately 20 days, the basal disk cells would be completely replaced.
    3. The conclusion that tentacle cells transdifferentiate into basal disc cells in the Zic4 knockdown may require more nuance, as only the tips of the tentacles express peroxidase. Do the more proximal regions of the tentacle express peduncle markers?

    Specific Points:

    Figure 1A, Figure 4E: The pictorial representation of Zic4 expression may need to be revised, as in situ hybridization data from Vogg et al., 2022, suggests that Zic4 is absent from the hypostome and tentacle tips. While in situ hybridization can sometimes lack precision due to variability in staining protocols and subjective decisions on when to stop the reaction, this observation aligns with scRNA-seq data, which also indicates a lack of Zic4 expression in the hypostome and tips of the tentacles.

    Figure 1 Legend: For panel D, the legend says "data taken from 28" but the references are not numbered. Same problem for panel E legend.

    Figure 1D: There may be a mistake in the Hydra body part labeling. Is "B" supposed to be "P" for peduncle?

    Figure 1 Panel E: Please provide clarification regarding what each box means. Are these 8 replicates of the same condition, or are these the proximal and distal regions of the tentacles as was collected in the Vogg paper?

    Figure 2A: Consider using the co-expression stats from Fig S2, which are very informative.

    Figure 2E, F: It would be more intuitive to group each experimental sample with its corresponding control.

    Figure 2C-F: Consider conducting statistical tests of significance between control and treatment groups.

    Figure 2 E - Considering the error bars, Gata3 upregulation in response to Zic4 knockdown does not look significant based on qPCR. Showing the significance of the up-regulation in the RNA-seq data may be more convincing. (I believe RNA-seq to be more reliable anyway).

    Figure S2: Might be helpful to show co-expression UMAPs here, like what is shown in Figure 2A.

    Page 4: "Interestingly, a similar binary choice pattern appears in certain neuronal lineages as well. A recent study demonstrated the involvement of Gata3 in specifying neurons at the aboral end (Primack et al. 2023), suggesting that this cross-regulation between Zic4 and Gata3 may extend beyond the epidermal lineage." Just a note that this paper shows expression, but doesn't show function as the statement implies, so the statement should be changed accordingly.

    Page 10: "Transcription Factor Binding site analysis... Hydra promoter sequences were compiled from the NCBI Hydra RP 105 assembly." Authors should provide a repository identifier for the genome they are using. Based on the information provided, it appears the authors are using Genome assembly "Hydra_RP_1.0" RefSeq GCF_000004095.1. However, that genome assembly has been suppressed for the following reason: "superseded by newer assembly for species". Authors should consider updating the reference assembly they are using to map their sequencing data and identify promoter sequences.

    The paper makes great use of the Hydra scRNA-seq data set! Minor point, when referring to the Hydra scRNA-seq data set, please cite Siebert et al., 2019 (data collection) and Cazet et al., 2023 (analysis that is being used in this paper).

    Something to keep in mind: To an audience without expertise in Hydra cell type morphology, the nematocyte marker HCR will likely be more convincing than the actin staining in Figure 3D to identify and quantify nematocytes.

    Minor Wording Issues:

    Page 2. "However, the mechanism by which Zic4 prevents the battery cell program from misexpression in normal tentacles remained unclear." Could read more clearly as: However, the mechanism by which Zic4 prevents the misexpression of the battery cell program in normal tentacles remained unclear.

    Page 2. "Potential candidates for this function could be found among TFs with highly enriched binding sites in the dataset, which are themselves Zic4 targets." Could read more clearly as: We reasoned that this intermediary factor, likely a target of Zic4, would be a transcription factor with highly enriched binding sites in the dataset.

    p3-4. "Q-PCR performed on dissected oral and aboral body regions confirmed this finding (Fig. 2C-D)" It is unclear which "finding" is being confirmed.

    Significance

    This compelling study from the Tsiairis lab uncovers a double-negative feedback loop between the transcription factors Zic4 and Gata3, functioning as a toggle switch to control oral and aboral fates in Hydra's epidermal lineage. Addressing fundamental questions in developmental biology, this research sheds light on the mechanisms underlying cell fate determination in relationship to their spatial organization. In Hydra, Wnt signaling, a conserved pathway critical for establishing primary body axes, promotes oral fate, emanating from an organizer at the oral end. Hydra body column epidermal cells can differentiate into distinct cell types, including oral battery cells and aboral basal disk cells, but the regulatory mechanisms remained elusive. Recent research from the Tsiairis lab identified Zic4 as a direct Wnt signaling target necessary for repressing basal disk-specific genes. Knocking down Zic4 caused battery cells to transform into basal disk cells, though Zic4 did not directly activate basal disk-specific genes, pointing to an intermediary regulator. This study identifies Gata3 as a key regulator of basal disk gene expression, as it is highly expressed at the aboral end, is inversely correlated with Zic4, and is upregulated in Zic4 knockouts. Functional experiments revealed mutual inhibition between Zic4 and Gata3: knocking down Gata3 led to differentiation of battery cells at the aboral end, while simultaneous knockdowns of Zic4 and Gata3 rescued the phenotypes of individual knockdowns. These findings demonstrate a finely tuned balance between Zic4 and Gata3 in regulating cell fate along the oral-aboral axis in Hydra. This paper therefore offers new insights into the spatial organization of cell type specification in Hydra and into broader principles of cell fate determination.

  5. Version published to 10.1101/2024.12.10.627691 on bioRxiv