Dear Dr Guigo,

Thank you very much for submitting your Research Article entitled 'bsAS, an antisense long non-coding RNA, essential for correct wing development through regulation of blistered/DSRF isoform usage' to PLOS Genetics. Your manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important problem as well as the novelty of the findings. However, both reviewers raised substantial concerns about the current manuscript. Based on the reviews, we will not be able to accept this version of the manuscript, but we would be willing to review a much-revised version. We cannot, of course, promise publication at that time.

Should you decide to revise the manuscript for further consideration here, your revisions should address the specific points made by each reviewer. We will also require a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript.

If you decide to revise the manuscript for further consideration at PLOS Genetics, please aim to resubmit within the next 60 days, unless it will take extra time to address the concerns of the reviewers, in which case we would appreciate an expected resubmission date by email to plosgenetics@plos.org.

If present, accompanying reviewer attachments are included with this email; please notify the journal office if any appear to be missing. They will also be available for download from the link below. You can use this link to log into the system when you are ready to submit a revised version, having first consulted our Submission Checklist.

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see our guidelines.

Please be aware that our data availability policy requires that all numerical data underlying graphs or summary statistics are included with the submission, and you will need to provide this upon resubmission if not already present. In addition, we do not permit the inclusion of phrases such as "data not shown" or "unpublished results" in manuscripts. All points should be backed up by data provided with the submission.

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool.  PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

PLOS has incorporated Similarity Check, powered by iThenticate, into its journal-wide submission system in order to screen submitted content for originality before publication. Each PLOS journal undertakes screening on a proportion of submitted articles. You will be contacted if needed following the screening process.

To resubmit, use the link below and 'Revise Submission' in the 'Submissions Needing Revision' folder.

[LINK]

We are sorry that we cannot be more positive about your manuscript at this stage. Please do not hesitate to contact us if you have any concerns or questions.

Yours sincerely,

Claude Desplan

Associate Editor

PLOS Genetics

Gregory P. Copenhaver

Editor-in-Chief

PLOS Genetics

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: In this work, Lluch and colabs searched for Natural Antisense Transcripts that could have a function during development by strand-specific RNA seq in different tissues of the fly. The authors identified bsAS as an antisense lncRNA to the bs/DSRF gene. bs is requiered for the proper vein-intervein pattern in the wing, trachea formation and also is implicated in neural processes such as memory, vision and sleep. Two different issoforms are described for bs. A long isoform (encoded by the A and C transcripts) and a short one (B transcript), being the later the one expressed in the wing disc. Interestingly, the authors beautifully demostrated that bsAS is expressed in the same intervein pattern as bs in the wing and that bsAS is requiered in cis to determine wich isoform is expressed. Mutants for bsAS showed a strong derepression of the long isoform without affecting the expression of the short one. Comparing the wing phenotypes of the bsAs mutants with the ectopic expression of the long isoform, the authors proposed that the intervein or neural cell fates are specified by the different ussage of the bs isoforms. Moreover, using 3C assays demostrated a physical contact between the transcription start site of bsAs and of the short isoform, suggesting a co-regulation. In addition, the authors perform a evolutionary analysis to propose a common origin of the bsAS and the long isoform structure of the bs gene.

The paper is well organized, written and the conclussions are novel and mostly well supported by their experiments. However, I have some discrepancies with some of the conclusions drawn from their results and few comments and questions that the authors may consider before publication.

1) One of my main problems with their model is that the authors conclude that the expression of the long isoform induces tissue neuralization (for example Fig. 5). In the text the authors mistake the vein as a neural tissue misleading the reader in several paragraphs in the text (pg. 17 for example). Also, the long isoform is normally not expressed in the wing and therefore, unless it is proven, is not requiered for vein patterning. Is it reasonable to think, as suggested by the authors, that the different functions of bs are carried out by the different isoforms, however this hypothesis was not tested in the paper.

A simple way to interpret their results is that the expression of the long isoform in the bsAS mutants acts as a dominant negative version of the short isoform, generating a partial bs loss of function phenotype and the formation of extraveins. Maybe looking at vein patterning genes in the different mutant combinations could help understand the phenotypes and function of the different isoforms. Also, I think is important to show the expression of bs, of the two different isoforms and of bsAS in the different tissues such as wing vs eye –disc.

2) In the discussion the authors “hypothesize that bs short isoform can counteract the expression of the long isoforms in the intervein regions, silencing the expression of neural genes and avoiding the development of ectopic veins within intervein regions”. However, if I understood correctly the paper and their results, is not the short isoform, but the transcription of the bsAS lncRNA the responsible for the downregulation of the long isoform in the intervein region. Again, the role of the long isoform inducing neural genes and ectopic vein development is not convincingly demosntrated.

It is a little confussing to me the different uses of isoform A or C in figure 5. They have shown that neither isoform are expressed in the wild type wing and only in the bsAS mutants, the isoform C is derepressed (pg. 14). So why picture iso A in the vein territories in fig. 5?

3) The upregulation of bs expression in the bsAS mutant is not evident at the RNA level in Fig. 2 and at the protein level it is hard to convince the readers comparing two different discs without any quantification. If the authors are convinced that there is an upregulation of Bs protein, maybe a WB could be more informative and quantitative.

Also, it is possible that the upregulation of the total bs levels is not detected because the derepression of the long isoform is very low compared to the short one, and therefore even an upregulation of the long one maybe masked and difficult to detect. I think this has to be clarified better.

4) In a bs mutant the entire intervein domain is transformed into vein tissue (see Fig. 2C Roch et al, 1998). This phenotype is different from what is observed in the homozygoes mutant for bsAS where some extra vein tissue is observed but most of the intervein region is preserved. I don't think the phenotypes are equivalent because in the bsAS mutant the short isoform is still normally expressed. The authors conclude that the bsAS mutant phenotype is a consequence of the overexpression of the long isoform. To demonstrate this important conclusion the authors could use specific RNAis available to the long isoforms such as JF02319 and KK108659 in the bsAS mutant background to test if the bsAS mutant phenotype could be restore.

5) Do the authors find any vein promoting gene upregulated in their RNA seq of bsAS mutant wings?. How is the pattern of genes such Knirps (kni) and Iroquois (Iro) in bsAS mutants?.

6) The authors showed a co-regulation of bsAS and the TSS1 using 3C techniques. As both bs and bsAS are expressed in the same intervein pattern, are they regulated by the same cis-regulatory module?. Is there a physical interaction between those regions with the identified wing CRM of bs (Nussbaumer et al, 2000) ?

Minor comments:

-p Value in Figure 1D and 2B for CG4812.

-is bs expressed in the EA disc? Maybe including a picture could be informative.

-The comparation and p values are not clear in Fig. 3C and D.

-Wl3 track for bs expression in Fig. Sup2I. The expression of the bs isforms is not observed. Compare it with Fig. 1C. Why?

Reviewer #2: In this study, Perez-Lluch et al., investigate the role of an antisense long non-coding RNA (lncRNA) in regulating usage of the bs/DSRF transcripts and the subsequent role in wing development. First, they describe the characterization of lncRNAs. First they identify the bs/DSRF gene as a potential candidate for transcript usage depending on lncRNA. Comparison of bs/DSRF transcript usage reveal strong differences in wing versus leg or eye-antennal discs. bs/DSRF has been shown to be expressed in the presumptive intervein tissue of the wing disc and the antisense transcript (bsAS) appears to be expressed in a similar pattern. Using a CRISPR/CAS9 strategy they make a specific deletion of the bsAS. This deletion results in the strong increase in the expression of the bs/DSRF long transcripts and in severe wing phenotype. The authors interpret this phenotype as a consequence of high bs/DSRF long transcript; however when over-expressing the bs/DSRF long transcript they observe minor (Fig 2H) or no visible phenotype (Fig S2E-F). Next, they show direct interactions between the transcription start site of bs and bsAS. Finally, the author analyze throughout evolution the conservation of the dual transcripts of the bs/DSRF orthologues. Experiments are properly performed to address the issue of the bsAS function. However there a major problem with the interpretation. The bsAS deficiency induces an extreme phenotype that is not at all comparable to the one induced by over-expression of the bs/DSRF long transcript. Thus, they cannot postulate that the extreme wing phenotype is due to the increased expression of the bs/DSRF long isoform. The conclusion (p14 bottom) and the model (Fig5) are over-estimated. I doubt that this study can be published in PLoS Genetics, unless they provide a consistent explanation based on experimental data for the severe wing phenotype observed in bsAS deficiency.

Additional comments:

1. Ref13 (Montagne et al., 1996) should be mentioned as the same time as ref10 (Fristrom et al., 1994) since ref13 is the one showing that bs and DSRF are allelic.

2. References for fly strains in M&M must be provided.

3. In situ hybridization in wing disc for bsAS and bs are not the “same’, they exhibit an overlapping pattern. The text should be modified.

4. The author cannot compare Fig2C (immunostaining to bs/DSRF) with Fig1E (in situ hybridization). Actually, there is no control for the genuine protein level in wild type disc in Fig2C.

5. bs/DSRF protein levels should be compared in the same experiment for wild type, for bsAS deficient and for long transcript-expressing wing discs.

6. The phenotype of bsAS heterozygous deficiency is minor, nonetheless, resembles the one of bs/DSRF long transcript over-expression; the author should also compare bs/DSRF protein levels in both genetic context.

**********

Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: Yes

**********

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

* Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. *

Dear Dr Guigo,

Thank you very much for submitting your Research Article entitled 'bsAS, an antisense long non-coding RNA, essential for correct wing development through regulation of blistered/DSRF isoform usage' to PLOS Genetics. Your manuscript was evaluated by the two initial independent peer reviewers. The reviewers found the paper much improved but still asked for you to address a few remaining points before the paper can be accepted. This should mostly require editorial changes

We therefore ask you to modify the manuscript according to the review recommendations before we can consider your manuscript for acceptance. Your revisions should address the specific points made by each reviewer.

In addition we ask that you:

1) Provide a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript.

2) Upload a Striking Image with a corresponding caption to accompany your manuscript if one is available (either a new image or an existing one from within your manuscript). If this image is judged to be suitable, it may be featured on our website. Images should ideally be high resolution, eye-catching, single panel square images. For examples, please browse our archive. If your image is from someone other than yourself, please ensure that the artist has read and agreed to the terms and conditions of the Creative Commons Attribution License. Note: we cannot publish copyrighted images.

We hope to receive your revised manuscript within the next 30 days. If you anticipate any delay in its return, we would ask you to let us know the expected resubmission date by email to plosgenetics@plos.org.

If present, accompanying reviewer attachments should be included with this email; please notify the journal office if any appear to be missing. They will also be available for download from the link below. You can use this link to log into the system when you are ready to submit a revised version, having first consulted our Submission Checklist.

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

Please be aware that our data availability policy requires that all numerical data underlying graphs or summary statistics are included with the submission, and you will need to provide this upon resubmission if not already present. In addition, we do not permit the inclusion of phrases such as "data not shown" or "unpublished results" in manuscripts. All points should be backed up by data provided with the submission.

PLOS has incorporated Similarity Check, powered by iThenticate, into its journal-wide submission system in order to screen submitted content for originality before publication. Each PLOS journal undertakes screening on a proportion of submitted articles. You will be contacted if needed following the screening process.

To resubmit, you will need to go to the link below and 'Revise Submission' in the 'Submissions Needing Revision' folder.

[LINK]

Please let us know if you have any questions while making these revisions.

Yours sincerely,

Claude

Claude Desplan

Associate Editor

PLOS Genetics

Gregory P. Copenhaver

Editor-in-Chief

PLOS Genetics

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: I acknowledge the effort and the experiments done by the authors to respond to the reviewers comments and suggestions. Overall, I really like the paper and it deserves to be published in PloS Genetics. The experiments proven the role of the LncRNA bsAS as a regulator of bs isoform usage are very solid.

However, I still identify some problems with the interpretation of the data that were raised in the first revision. Specifically, I find the the role of the bs long isoform as a determinant of vein fate during normal development not been proved. Moreover, the authors insists in consider the vein tissue as a neural tissue and in my opinion this is an inaccuracy and could lead to confussion.

To prove the role of the bs long isoform as a determinant of vein tissue in the wing, the authors should demonstrate:

-1) bs long isform expression in the vein region. In situ hybridization of the bs long isoform?

-2) the funtion of the bs long isform in a wt background in the wing. A simple experiment would be to knockdown the bs long in the wing with an specific RNAi. If their model (fig. 6) is correct, the prediction would be wings with defective veins. However, in fig. 2J (although no ideal as it is performed in the bs+/- background) no defect on the characteristic vein pattern is observed.

Sentences like “In contrast, in the vein regions, in which bsAS is poorly expressed, the long isoforms (A or C, or both) are the dominant ones, inducing the expression of neural genes and the differentiation of veins” and their model in fig. 6 lead to a function of the long isform as determinant of vein fate that has not been proven.

The authors also include a new experiment where cells GFP+ and GFP- cells are isolated of third instar larvae wings of bs-GAL4>UAS-GFP flies and conclude that the bsAS and short isoforms are expressed mainly in intervein (GFP+) while long isoforms are expressed at similar levels in vein and intervein. GFP- cells are formed by vein cells (minority) and hinge and notum cells (mayority), and therefore this experiment is not very precise, informative and conclusive.

Reviewer #2: The revised version of the manuscript of Perez-Lluch et al., is more convincing than the first one. My main concerns have been addressed; in particular, immunostaining pictures that strengthen the relationship between bsAS activity and bs protein level expression, and novel data showing that selective knockdown of the bs/DSFR long isoform can significantly suppress the wing phenotype of bsAS-/- mutants.

Minor point: In the discussion, the authors stress the ancestral conservation of bs and bsAS throughout evolution, even in wingless animals, and conclude by an original function in neural differentiation. However, wings have been proposed to have evolved from ancestral gills (doi.org/10.1038/385627a0). Further, dSRF plays a critical role in the differentiation of the terminal tracheal cells (Guillemin et al 1996 Development 122:1353-62). Therefore, one can also speculates that the ancestral conservation of bs and bsAS may rely on an original function in the development of the respiratory system. Although it would be easy to look at the differentiation of the terminal tracheal cells in bsAS mutant flies, I consider that it is not the purpose of the present study. Nonetheless, I suggest a modulation of the neural differentiation origin in the conclusion.

**********

Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: Yes

**********

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Dear Dr Guigo,

We are pleased to inform you that your manuscript entitled "bsAS, an antisense long non-coding RNA, essential for correct wing development through regulation of blistered/DSRF isoform usage" has been editorially accepted for publication in PLOS Genetics. Congratulations!

Before your submission can be formally accepted and sent to production you will need to complete our formatting changes, which you will receive in a follow up email. Please be aware that it may take several days for you to receive this email; during this time no action is required by you. Please note: the accept date on your published article will reflect the date of this provisional accept, but your manuscript will not be scheduled for publication until the required changes have been made.

Once your paper is formally accepted, an uncorrected proof of your manuscript will be published online ahead of the final version, unless you’ve already opted out via the online submission form. If, for any reason, you do not want an earlier version of your manuscript published online or are unsure if you have already indicated as such, please let the journal staff know immediately at plosgenetics@plos.org.

In the meantime, please log into Editorial Manager at https://www.editorialmanager.com/pgenetics/, click the "Update My Information" link at the top of the page, and update your user information to ensure an efficient production and billing process. Note that PLOS requires an ORCID iD for all corresponding authors. Therefore, please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field.  This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager.

If you have a press-related query, or would like to know about one way to make your underlying data available (as you will be aware, this is required for publication), please see the end of this email. If your institution or institutions have a press office, please notify them about your upcoming article at this point, to enable them to help maximise its impact. Inform journal staff as soon as possible if you are preparing a press release for your article and need a publication date.

Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Genetics!

Yours sincerely,

Claude Desplan

Associate Editor

PLOS Genetics

Gregory P. Copenhaver

Editor-in-Chief

PLOS Genetics

www.plosgenetics.org

Twitter: @PLOSGenetics

----------------------------------------------------

Comments from the reviewers (if applicable):

----------------------------------------------------

Data Deposition

If you have submitted a Research Article or Front Matter that has associated data that are not suitable for deposition in a subject-specific public repository (such as GenBank or ArrayExpress), one way to make that data available is to deposit it in the Dryad Digital Repository. As you may recall, we ask all authors to agree to make data available; this is one way to achieve that. A full list of recommended repositories can be found on our website.

The following link will take you to the Dryad record for your article, so you won't have to re‐enter its bibliographic information, and can upload your files directly: 

http://datadryad.org/submit?journalID=pgenetics&manu=PGENETICS-D-20-00125R2

More information about depositing data in Dryad is available at http://www.datadryad.org/depositing. If you experience any difficulties in submitting your data, please contact help@datadryad.org for support.

Additionally, please be aware that our data availability policy requires that all numerical data underlying display items are included with the submission, and you will need to provide this before we can formally accept your manuscript, if not already present.

----------------------------------------------------

Press Queries

If you or your institution will be preparing press materials for this manuscript, or if you need to know your paper's publication date for media purposes, please inform the journal staff as soon as possible so that your submission can be scheduled accordingly. Your manuscript will remain under a strict press embargo until the publication date and time. This means an early version of your manuscript will not be published ahead of your final version. PLOS Genetics may also choose to issue a press release for your article. If there's anything the journal should know or you'd like more information, please get in touch via plosgenetics@plos.org.