Crosstalk between nitric oxide and retinoic acid pathways is essential for amphioxus pharynx development

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1. 

   Version published to 10.7554/elife.58295 on eLife

   Aug 25, 2021
2. 

   eLife

   Jun 24, 2020

   ###This manuscript is in revision at eLife

   The decision letter after peer review, sent to the authors on June 12, 2020, follows.

   Summary

   All three reviewers agree that the research question under study, the requirement of the cross-talk between two important developmental signaling pathways - retinoic acid and the NO - for amphioxus pharynx development, is in principle interesting and could be suitable for publication in eLife.

   However, at present there are major open concerns especially on the lack of statistical analyses, quality of data presentation and inconsistencies with previously published work, that need to be addressed. Although it is the current policy of eLife to avoid additional experiments in revisions as much as possible, this is unfortunately likely impossible to fulfil with the current manuscript in order to bring it to …

   ###This manuscript is in revision at eLife

   The decision letter after peer review, sent to the authors on June 12, 2020, follows.

   Summary

   All three reviewers agree that the research question under study, the requirement of the cross-talk between two important developmental signaling pathways - retinoic acid and the NO - for amphioxus pharynx development, is in principle interesting and could be suitable for publication in eLife.

   However, at present there are major open concerns especially on the lack of statistical analyses, quality of data presentation and inconsistencies with previously published work, that need to be addressed. Although it is the current policy of eLife to avoid additional experiments in revisions as much as possible, this is unfortunately likely impossible to fulfil with the current manuscript in order to bring it to a level that matches the standards of eLife. However, we think that in many cases an improvement of analyses and data presentation will likely already significantly improve the manuscript.

   1. The presented study is a follow-up on a previous paper by the same lab (Annona et al 2017 ; DOI:10.1038/s41598-017-08157-w). When comparing the work of this previous study with the current manuscript two major discrepancies are apparent:

   In Annona et al the two drugs were used to inhibit NOS production: L-NAME and TRIM, while only one inhibitor was used in the present study. Furthermore, there appear to be discrepancies concerning the developmental time windows during which chemical disruption of NO signaling is effective described in the two publications. This needs to be clarified.

   The timing of NosA,B,C expression, the suggested regulation of NosA and B by retinoic acid (RA) and the detected presumptive RARE regulatory elements in the genome don't match. More specifically, NosA,B,C expression at 24 hours (or around this time point) was investigated by Annona et al, 2017. Based on these data, NosA is not expressed during development, whereas NosB and NosC are expressed. In the submitted manuscript, the authors show that NosA and NosB are upregulated upon RA treatment, whereas NosC shows no changes in expression. They therefore suggest that RA regulates NosA and NosB transcription. Since only NosB is expressed during the relevant timepoints at early development, the transcription of this gene could be under the regulation of RA. However, when the authors look into the retinoic acid response elements (RARE) in the genomic region of NosB, they only find a DR3, which is not the typical RARE. They find DR1 and DR5 (apart from DR3's), which are more typical RARE's, in the genomic region of NosA, but as mentioned this gene is not expressed during development. This makes the hypothesis of a direct regulation of NosA and NosB by RA during normal development unconvincing. Can the author dissolve these apparent discrepancies?

   1. The authors study the open chomatin structure at 8, 15, 36 and 60 hours, thus time points, which do not overlap with the drug treatment period (24-30 hours). They need to analyze the genome architecture at this time period.

   2. The previous work by Annona et al 2017 et al shows that a major peak at NO levels occurs later than the chosen treatment window. How do NO levels during the time window of the experiment compare with other studies, i.e. is there evidence these are relevant levels? This is particularly noteworthy, as there is no control experiment showing that TRIM incubation affects NO levels or NO signaling during the incubation period (e.g. DAF-FM-DA staining or by NO quantification). It is therefore not possible to estimate the specificity of the resulting phenotypes.

   We thus request from the authors to provide ISH patterns of all the Nos genes, as well as NO localisation from at least 2 timepoints (e.g. start and end of window) of the TRIM application window.

   1. One overarching critique is that the general description of the figures and hence also the phenotypes are of poor quality. An improvement of this point will already majorly improve the entire manuscript.

   Fig.1A: Indicate developmental stages (N2, N4, T1, T2, T3, L0) together with the hours-post-fertilization (hpf) to facilitate the understanding of the treatment period with respect to the development of amphioxus.

   Fig.1B: Outline pharyngeal region e.g. with thin, dashed white lines in longitudinal and cross-sections and indicate relevant anatomical structures (club-shaped gland, endostyle, gill slits) e.g. with an arrow. Is the endostyle positioned more ventrally in TRIM treated larva?

   Figure 1C: why are Cyp26.3, Rdh11/12.18 and Crabp shown in triplicates?

   Fig.1B: The 'digital sectioning' method using confocal imaging and reconstruction of nuclear stainings is not suited to characterize the phenotype. Due to the loss of signal in deeper regions, morphological structures (e.g. differences in pharyngeal and gill slit morphology, endostyl, club-shaped glands) are impossible to recognize.

   Fig.3B: the heads of these amphioxus should be annotated to indicate key structures for non-amphioxus specialists. Ideally the images should be higher magnification and resolution as well, as the morphology is currently not very clear.

   Fig.3A and B: Furthermore, the morphological differences between 'altered', 'partially recovered' and 'recovered' is unclear. Fig.3B does not help understanding changes as the pictures are too small to recognize any morphological details without staining, and no structures are indicated. It is also unclear how animals scored as 'altered', 'partially recovered' and 'recovered' differ in their morphological structures. And does 'recovered' mean that these embryos show an initial phenotype that then 'recovers' during development, or do they show a completely normal development?

   1. Missing statistics/statistical information: Lines 85-89 (Fig.1): Where is the evidence that there is reduction in pharynx length? Where is the evidence for a smaller first gill slit? Measurements with a decent sample size and a basic statistical test must be provided.

   The description of ISH pictures in Fig.2A lacks any quantification and thus any information on the penetrance of the respective phenotypes are (as in Fig 3C). The lack of any 'negative control genes' (the large set of genes that, based on the RNASeq dataset, should not be affected) make it difficult to judge how specific changes in AP axis and RA pathway genes are.

   How did the authors obtain the qRT-PCR calculations? They need to clarify how they obtained the Fold changes shown in the histograms .e.g. by showing the maths behind the result when marking the cells in the excel sheet. The raw data for rpl32 is missing for Crabp in Figure 2B. The qPCR results in Fig.2B-E lack significance tests.

   1. The RNA-Seq study needs improvements: The PCA (Fig.S1C) shows no concordance among control samples or treated samples. Also, the histogram shows a clustering of replicates, and NOT of 'treated' and 'control' samples. This casts doubts on the quality and validity of the RNASeq dataset. These doubts are not removed by the current validation experiments, as these experiments tested only significantly upregulated genes by RNA-Seq, while downregulated and non-significant genes as 'controls' are missing. These additional controls are necessary to assess the validity of the RNA-Seq data.

   2. More information about the details of the ATACseq and ChIPseq data used, as well as the general RA responsive elements prediction is required.

   For example, in what amphioxus samples (and treatments if any) are these ATACseq and ChIPseq signals seen? There is some detail provided in the Methods section, but something is odd here and perhaps needs some further explanation. Since the two relevant Nos genes are supposedly not active during development then why do they have ATACseq and ChIPseq signals from embryo and larval samples? Why should these two Nos genes have apparently active regulatory elements focused on RAREs when the genes are not normally expressed under the control of RA, but only become active when exogenous RA is applied? We may well have missed something in the logic here, but this merely shows that the current level of explanation is insufficient.

   The analysis of RA responsive elements lacks statistical analysis and depth. It is left unclear how many RAREs would be expected by chance on a 52kb resp. 25kb locus. In addition, the authors include all ATAC-Seq peaks from stages ranging between 8h and 60hpf, while the window of RA responsiveness has been tightly restricted to the 24h-30hpf window. Also, as NosC expression levels stay constant upon RA incubation, it would be crucial to know if the NosC locus lacks any open RARE sites (as would be expected).

   The authors use NHR-SCAN tool to predict putative direct repeats binding sites in the genomic sequence of NosA and NosB. Which consensus sequence does the program follow? It appears that it does not follow the consensus sequence for typical RARE ((A/G)G(G/T)TCA), since the sequence for DR1 deviate from this sequence? DR1, DR2 and DR5 are the commonly described binding RARE's for the RAR/RXR heterodimers. Further, DR8 has been described as retinoic acid dependent regulation of gene transcription through RAR/RXR (Moutier et al., 2012). The authors need to provide clarification which are the most commonly used RARE's of the DR's detected.

   Please also mention if RAREs fall within an intron in the genomic regions of the Nos genes, since the transcriptional regulation through RARE is often associated to introns.

   1. Information on the concentration dependency of compounds used in the rescue experiment is lacking. Please explain why the BMS009 concentration used here (10exp-6 M) is 10x higher than the highest concentration used in the original publication on amphioxus pharynx development (Escriva et al., Development 2002).

   2. A summary drawing of the regulatory loop between NO and RA would be informative, also indicating the known target genes (from this study).

   Read the original source
3. 

   Version published to 10.1101/2020.06.22.164632 on bioRxiv

   Jun 23, 2020