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    Referee #4

    Evidence, reproducibility and clarity

    Summary:

    The freshwater polyp Hydra possess the remarkable ability to regenerate a fully functional head within a few days after amputation, however when e.g., Notch signaling is inhibited the animals fail to regenerate the original head pattern. In the manuscript by Moneer et al. the authors aim to identify Notch responsive genes by RNA sequencing. 48 hours after Notch signaling inhibition with DAPT, 624 genes were up- and 207 genes downregulated. To identify putative direct Notch target genes the authors generated RNA-seq datasets at 3 and 6 hours after DAPT removal and propose that the expression of direct target genes is rapidly recovered within 3 hours as shown by the re-expression of HyHES. Furthermore, by performing motif enrichment analyses the authors propose that e.g., HyAlx and HySp5 could be direct Notch target genes.

    Major comments:

    1. It is not clear why the authors chose 48 hours as a time point for RNA sequencing. Why not 12 or 24 hours after DAPT exposure? Is the expression of HyHES or CnASH not downregulated at earlier time points? Furthermore, why did the authors use whole animals and not just the head tissue for RNA-seq to enrich the transcripts?

    2. Why did the authors not perform RNA sequencing on head regenerating DAPT-treated animals? This would help to better understand the relationship between Notch and Wnt signaling especially as the authors showed in 2013 (Mündner et al) that the expression of Wnt3 is strongly affected in head regenerating DAPT-treated animals.

    3. It is currently very difficult to fully evaluate the data. One single excel file with all up- and downregulated candidates should be provided (Trinity ID, fold change, False Discovery Rate, annotation etc.). I would have assumed that genes such as Wnt8 that are expressed at the base of the tentacles (Philipp et al., 2009) could be affected by DAPT. Is Wnt3 not affected at all in intact animals?

    4. The silencing of Sp5 induces the formation of ectopic heads in intact and regenerating conditions and it has clearly been shown that Sp5 inhibits Wnt/β-catenin signaling. To call Sp5 a tentacle patterning gene just based on the identification of RBPJ-motifs in the Sp5 regulatory region is misleading, as it is currently not supported by experimental data. The fact that a regulatory motif is present in a promoter region does not mean that this regulatory motif is active.

    5. This manuscript would be much more interesting and of greater importance if the authors would have added functional data for one or two candidate genes.

    Minor comments:

    1. Figure S1: Individual data points for the qPCR analysis should be shown and arrow bars added.

    2. Figure 6: Scale bars are missing.

    Significance

    The manuscript is well written, and the presented results could be of interest for the Hydra field but they will not have a broad impact in the present state. I find it unfortunate that the authors did not use the datasets produced to better understand the complex regulatory network that is active during the patterning of the Hydra head.

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    Referee #3

    Evidence, reproducibility and clarity

    Moneer et al. studied Notch target genes in the context of nematogenesis, i.e. the generation of stinging cells (nematocytes) from interstitial stem cells (i-cells), and in axial patterning, in the cnidarian Hydra. They used the Notch pathway inhibitor DAPT, a drug acting on presenilin, preventing the release of Notch intracellular domain (NICD). Bottger's team pioneered the usage of DAPT in Hydra back in 2007 and it has been used successfully since then in other cnidarians too. The authors first exposed Hydra polyps to DAPT for 48 hours, followed by transcriptomic analysis to identify Notch responsive genes. They then analyzed gene expression at 3 and 6 hours after removal of DAPT to identify direct and indirect Notch targets, respectively. Using a recently published Hydra single-cell atlas, the authors report that most Notch responsive genes are expressed in the nematocyte lineage, consistent with the known role of Notch signaling in hydrozoan nematogenesis. They also identify Notch targets in epithelial cells, consistent with a role of the pathway in axis patterning.

    Overall, the manuscript is interesting, and the authors' conclusions are overall supported by the data. A strength of the paper is the good usage they make of a previously published Hydra single-cell transcriptome, which they do in collaboration with the Juliano lab who generated this data set. A weakness of the work is the dependence on Notch pharmacological inhibition and absence of genetic interference; the latter would provide evidence for specificity as opposed to phenotypes being a side effect of DAPT or high DMSO concentration (e.g. stress response, see specific point #6, below). The text reads well, and the figures are of good quality. Below is a list of points to be addressed.

    1. On p. 4, the authors state: "We identified 831 genes that were differentially expressed in response to 48 hours of DAPT treatments". This refers to genes differentially expressed at T0. Then, they check the expression of these genes at T3 and T6. Were all differentially expressed genes at T3 and T6 included in the 831 genes identified at T0? Did the authors find differentially expressed genes at T3 and T6 which are not differentially expressed at T0?

    2. p. 2, last paragraph: insert "the time points 3 and 6 hrs after DAPT removal" after "To characterize...". This is important to clarify that the analysis was done after removal rather than the addition of DAPT.

    3. The authors normalized the expression of genes of interest to several housekeeping genes (RPL13, SDH, EF1α, GAPDH, and Actin) in their qPCR analysis. In Fig. S1, however, only "control" is written. Did the authors merge all results from the different housekeeping genes, or did they use only one reference gene as control (which one?) to generate the figure?

    4. On Fig. 3 and the accompanying text on p 5, the black and grey clusters represent 90 and 80 genes, respectively. These 170 genes represent 25% of the total (170/666), not 20%. Clarify.

    5. The figure number of Figure S2 is not indicated in the figure.

    6. Can the authors confirm the DMSO concentration (1%)? I am aware this was the concentration used in their previous work, but it is nevertheless pretty high. High DMSO concentration could explain the stress response they observed.

    7. Figure 1: on the right, few letters are missing.

    8. Fig. 5B, remove lettering J,K,L from lower panel images.

    9. Figure number is absent in Figure 9.

    10. The authors completely ignore work on Notch signaling in other cnidarians. This not only impedes an evolutionary synthesis of the data but also leads to failure to discuss other functions Notch fulfills in cnidarian biology (e.g. immunity and regeneration).

    Significance

    The Notch pathway inhibitor, DAPT, has been widely used in work involving cnidarians. These studies have established a role for Notch in late-stage stinging cell differentiation and in tentacle morphogenesis in development and regeneration (Layden and Martindale, 2014; Marlow et al., 2012; Munder et al. 2013; Richards and Rentzsch, 2014, 2015; Gahan et al., 2017). It has also been shown that early-stage neurogenesis in hydrozoans is independent of Notch (Kasbauer et al. 2007; Gahan et al. 2017), which is different from bilaterian and anthozoan neurogenesis. What Moneer et al. did in the present study was to take these known phenotypes and put them in a cellular and molecular context. The results, showing that nematogenesis genes are Notch targets, are not surprising but novel. This work closes an existing knowledge gap and is important for the field.

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    Referee #2

    Evidence, reproducibility and clarity

    In the manuscript by Moneer et al. Notch target genes are defined in Hydra using a classical Gamma-secretase inhibition approach. Gene expression analyses is done at different time-points via RNAseq and combined with single-cell data and ATAC-seq data. This is further elaborated with exact expression analysis and experiments studying the wash-out (recovery) of the inhibitor and again gene expression profiling. Regarding the target genes identifies several new and interesting target genes. The downstream transcription factors Pou4F3 and Pax6 are very interesting and the Wnt-pathway regulators as well. This is way more convincing than the previously described cross-talks.

    My comments:

    1. Introduction (page-3): Only few direct target genes of Notch-signaling have been identified so far. I don't agree. By now, there are several studies in the mammalian system using ChIPseq with anti-RBPJ and GSI-studies and dnMAML followed by RNAseq. In addition, there is also genomic fairly good data using the Drosophila-system. (On the other hand, there is still a need to identify in better defined systems). Please correct and add additional references.

    2. Regarding Figure-2: How many genes are in each class? Are all the 624 genes downregulated after 48 hours of DAPT? (Part of these genes could still be direct Notch targets, possibly also harboring RBPJ binding motifs).

    1. Some of the genes in the mammalian systems do not appear in presented study in Hydra: What happens the feedback regulators Dtx and NRARP? Is the Hydra Notch-gene itself regulated? What about oncogene c-myc? (I assume that c-myc exists also in Hydra (?).

    2. Evolutionary conservation; (Regarding addition to Figure-9): For readers that are not so familiar with Hydra, it would be extremely helpful to have a summary-table (list) with conserved Notch target genes.

    1. Suggestion: I am not a Hydra-expert, but, if possible, experiments using inducible dominant-negative Mastermind (dnMAML) would strengthen this manuscript.

    Significance

    This study by Moneer et al. is a nice and thoroughly done study, which will further advance our understanding of Notch target genes. This is of interest of readers in signal transduction and developmental biology.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary

    The manuscript of Moneer et al describes RNAseq data on DAPT treated Hydra aiming to identify genes involved in the Notch pathway. The RNAseq data is compared with previously published Singe Cell Seq data. They proceed to perform hierarchical clustering, motif enrichment analysis of promoter regions and metagene analysis. The research provides a resource for other researchers that are interested in Notch signalling in Hydra.

    Major comments

    The research is very descriptive in nature. The RNAseq experiment is mostly well set up and analysed, however, the manuscript lacks subsequent experiments to confirm their findings or to determine the possible significance of the data. As a consequence, the authors are not able to draw clear conclusions from the data as most findings are only suggestive.

    The manuscript aims to identify Notch dependent molecular pathways. However, the authors find a lot of indirect targets and a lot of the analyses involve these targets. In comparison, the few potential direct targets, which should be the core of the manuscript, do not receive sufficient attention. The manuscript would be much more significant if the focus would be on the direct targets and would include experiments to determine if the suggestions the current data provides can be confirmed and expanded upon.

    Only two time points were used to establish which two time points were required to be able to differentiate between direct and indirect targets. This experiment requires more time points as well as several known direct and indirect targets as different targets will recover at different rates. Only then will the authors be able to determine whether they used the most appropriate time points.

    A significant number of the figures relies heavily on a previously published paper from the same group. The methods section lacks a description of the statistical analysis performed.

    Minor comments

    The title of the manuscript is too strong for the data provided.

    Although the introduction is well written, the results section lacks clarity and explanation. A concluding sentence at the end of each paragraph would aid the reader in analysing the significance of the findings. In results section 2 the authors mention the identification of 23 metagenes. A figure/table presenting this data would aid the presentation of this data. Fig 6 shows in situ hybridisation data that could potentially be interesting, however, the authors could add some more information to link this data to the Notch pathway.

    In Fig S1 information about the control is lacking. Fig S3 shows alignments and phylogenetic trees but it is not clear what the function is of this figure. Some additional information explaining the relevance of the data would improve the manuscript.

    In the methods section additional information regarding the set up and analysis of the qPCR is required (see MIQE guidelines). This includes further information on how the primers were tested.

    Several of the figures use colour coding but some of these are not defined in the legends. Some of the figures/tables use abbreviations that are not defined. References are split between the regular reference list and a separate list in table S2. There appear to be very few recent references.

    Significance

    The manuscript provides a potential resource for further research. It might be relevant to researchers interested in Notch signalling and/or Hydra as a model organism/evolutionary studies. The data is mostly descriptive in nature. To date Notch signalling in Hydra has not received a lot of attention in the existing literature. The reviewer's area of expertise is Notch signalling in development. The reviewer is not familiar with Hydra as a model system.

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