Ribbon boosts ribosomal protein gene expression to coordinate organ form and function
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Abstract
Cell growth is well defined for late (postembryonic) stages of development, but evidence for early (embryonic) cell growth during postmitotic morphogenesis is limited. Here, we report early cell growth as a key characteristic of tubulogenesis in the Drosophila embryonic salivary gland (SG) and trachea. A BTB/POZ domain nuclear factor, Ribbon (Rib), mediates this early cell growth. Rib binds the transcription start site of nearly every SG-expressed ribosomal protein gene (RPG) and is required for full expression of all RPGs tested. Rib binding to RPG promoters in vitro is weak and not sequence specific, suggesting that specificity is achieved through cofactor interactions. Accordingly, we demonstrate Rib’s ability to physically interact with each of the three known regulators of RPG transcription. Surprisingly, Rib-dependent early cell growth in another tubular organ, the embryonic trachea, is not mediated by direct RPG transcription. These findings support a model of early cell growth customized by transcriptional regulatory networks to coordinate organ form and function.
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Reply to the reviewers
Reviewer comments:
Reviewer #1 (Evidence, reproducibility and clarity (Required)):
In this paper, the authors examine the relationship between the transcription factor Ribbon, its ribosomal protein gene (RPG) targets, and cell growth during the process of salivary gland tubulogenesis in the Drosophila embryo. This study builds upon previous work they published in 2016 (Loganathan et al., 2016). While the previous study identified RPGs as potential targets of Ribbon from ChIP-Seq analysis, they did not delve into the role of these targets in salivary gland morphogenesis. Here, the authors demonstrate that mutation of ribbon results in decreased cell …
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Reply to the reviewers
Reviewer comments:
Reviewer #1 (Evidence, reproducibility and clarity (Required)):
In this paper, the authors examine the relationship between the transcription factor Ribbon, its ribosomal protein gene (RPG) targets, and cell growth during the process of salivary gland tubulogenesis in the Drosophila embryo. This study builds upon previous work they published in 2016 (Loganathan et al., 2016). While the previous study identified RPGs as potential targets of Ribbon from ChIP-Seq analysis, they did not delve into the role of these targets in salivary gland morphogenesis. Here, the authors demonstrate that mutation of ribbon results in decreased cell volumes via immunostaining and image analysis. They identify and confirm RPGs as ribbon transcriptional targets using ChIP-SEQ, Microarray data, in situ hybridization, and qRT-PCR. They analyze these targets in an effort to identify a Rib consensus binding sites by MEME and find that Rib binding is not specific using EMSA. They suggest specificity arises from association with transcriptional cofactors. Binding with cofactors was confirmed by CO-IP and in vivo RNAi experiments demonstrated the requirement of these cofactors in mediating changes in cell volume during salivary gland tubulogenesis. They demonstrate that Ribbon regulation of cell growth via transcription of RPGs is not a universal mechanism for Ribbon function, as Ribbon regulates transcription of other genes in the context of tracheal development.
**Major comments:**
Results of all experiments are conclusive, and significant numbers of samples were noted for most figure panels. For a few panels the sample number/number of replicates was not noted, and it is recommended that the authors add this information (Figure 1F; 5B,C; 7B).
Additional experiments are not needed to support the conclusions presented in this work. The data and methods are presented clearly and the statistical analyses performed were appropriate.
In regard to microarray data, Figure 4E shows fold change as log2 values, but it is unclear if this is the case for Table S2. This should be clarified. The authors note in the text on page 7 that few targets show a greater than 1.5-fold change. Based on Figure 4E, this is a log2 value, and should be specified as such.
As the Rib antibody was generated in this study, it would be helpful to include data illustrating a confirmation of antibody specificity. This could include Rib antibody staining on rib mutant embryos, or showing a lack of band for ribbon in ribbon mutants on a Western blot. If the specificity has been published elsewhere, please add a reference.
**Minor Comments:**
As the microarray data was previously published in Loganathan et al 2016, as mentioned in the results section, this citation should also be included in the Methods section describing the Microarray data.
In the discussion section on page 15, a list of factors in the gene network are listed. What is viz.?
Reviewer #1 (Significance (Required)):
•As described in the introduction, the role of cell growth during embryonic tissue morphogenesis is a relatively unexplored topic. The authors point out that most previous studies describing regulation of tissue growth have focused on the role of mitosis and increased polyploidy, as in the gut (https://doi.org/10.1016/S0925-4773(00)00512-8 ), as primary mechanisms. In the case of the salivary gland, only a single endocycle occurs during embryogenesis and cells are post-mitotic, suggesting another mechanism is at play. This study identifies Ribbon as a mediator of cell growth and demonstrates that Ribbon mediates this function through transcriptional regulation of RPGs. In addition, they identify Ribbon cofactors that are important for salivary gland cell growth and tissue morphogenesis. Interestingly, they find that this mechanism for cell growth may be tissue specific, as Ribbon appears to regulate different genes in the trachea.
•This work has implications for the regulation of cell growth in other tissues and organisms and would be of broad interest to those studying organ development.
•In order to contextualize my review, I am a developmental biologist that works with Drosophila.
**Referees cross-commenting**
In regard to the comments by reviewer #2: I agree that point # 2 should be addressed to more thoroughly describe the method, but as the authors have looked at DNA Amplification at a time point following the normal endocycle, which occurs at stage 12, and DNA content is not significantly different, I don't think analysis of earlier stages would influence their conclusions.
Given that the authors do include some RNAi data for RPGs and Trf2, it would enhance the paper further to include M1BP and Dref RNAi data if quality reagents are available as described in point 5. Point 6 can be easily addressed. In regard to point 8, the effects of rib overexpression alone would be interesting to see given the ability of this construct to rescue the phenotype.
While I think points 3 and 7 are excellent ideas for a follow up study, I think they are outside of the scope of this paper. I do not view point 4 as essential to this study, as the study focuses on the regulation of transcription of the RPGs by Rib.
In regard to the comments by reviewer #3, I agree that points 1 and 2 should be addressed. It would be extremely difficult to address point #3 by dissecting out the tissue, but it could be addressed via further explanation in the text, as could point #4. I don't think minor points 4-6 need to be addressed, but the minor points 1-3 should addressed to improve the paper. For minor point #3, I would suggest the number of genes be included in Supplementary Table 1.
As reviewer #1, I think my comments should be addressed to improve the quality and clarity of the paper.
Reviewer #2 (Evidence, reproducibility and clarity (Required)):
This paper reported a role for the BTB/POZ-domain transcription factor rib in mediating early cell growth of embryonic salivary gland (SG) cells. the authors show that during tubulogenesis of the salivary glands, rib binds the transcription start site of almost all SG-expressed ribosomal protein gene (RPG) and promotes their transcription, thus providing a material foundation for cell growth. Interestingly, in embryo trachea cells, rib targets do not include RPGs, which indicates that rib may use different mechanisms to regulate cell growth of different organs. In general, this is a well-written, well designed research article with many conclusions well-supported by experimental evidence. Listed below are a few issues (mostly minor/unessential) for the authors to consider.
**Major comments:**
1.Although in Figure 1G, the nucleus size is indistinct in rib mutant and wt cells at stage 15 and 16, Figure 1C appeared to look like that the rib mutant nuclei at stage 11, 13 and 14 are significantly smaller than those in wild type cells. The authors need to make sure that the rib phenotype has nothing to do with DNA amplification.
2.Please describe the details on calculating DNA volume by DAPI staining in the method session.
3.The authors have demonstrated weak DNA binding ability of Rib, and physical interactions between Rib with the known regulators of RPG transcription (Trf2, M1BP, and Dref), but what is the functional relationships between Rib and the known RPG regulators? e.g., does Rib function to promote DNA binding and transcriptional activity of Trf2, M1BP, and Dref, or vice versa?
4.To confirm the rib function on RPG translation, it is recommended to examine ribosomal proteins by western, and comparing the total protein content would also be helpful.
5.As Trf2, M1BP and Dref are physically interacted with Rib, it would be helpful to determine Whether M1BP and Dref knockdown can phenocopy the cell growth deficit observed in rib mutant SGs.
6.Page12, paragraph 3, "Thus, despite the shared requirement for Rib in embryonic cell growth of both tubular organs, Rib-dependent growth in the trachea is likely through regulation of alternative growth-promoting factors." Please list the potential growth-promoting factors targeted by Rib according to the Chip-seq data, if possible.
7.It would be interesting to determine whether rib mutation differently affect the secretory function of salivary gland at embryo, larva, pupa or adult stage.
8.Does Rib overexpression have any effects to SG development? Considering the authors adopted GAL4-UAS system to rescue Rib under Rib-KO, it would be interesting to see if Rib overexpression could cause an opposite overgrowth phenotype.
Reviewer #2 (Significance (Required)):
This paper discovered a new mechanism underlying organ-specific cell growth regulation during a specific time-window of animal development, which should be of interest to the field of cell and developmental biology.
Drosophila genetics; Developmental biology
**Referees cross-commenting**
I agree with all the other referees that the comments raised by reviewer #1 should be addressed entirely.
In regard to the comments by reviewer #3, all of the 4 major points are excellent and should be addressed, but it is okay to address points #3 and 4 by simple explanation or re-wording. I find the minor point #6 is nice to have but not essential, the rest should be addressed.
In case of my comments (reviewer 2), points #1,2,5,8 should be addressed, others are nice to have.
Reviewer #3 (Evidence, reproducibility and clarity (Required)):
In the manuscript "The Ribb-osome: Ribbon boosts ribosomal protein gene expression to coordinate organ form and function" the authors show evidence that Ribbon mediates early cell growth in Drosophila embryonic salivary gland through direct interaction with ribosomal protein genes. The manuscript is well written while presenting novel and solid data. The data could be strengthened by some further analysis and clarification, but none of the issues raised represent major flaws.
**Key points:**
1.Cell segmentations: The way the cell segmentations / volume quantifications are presented it is impossible to judge their quality. The authors should provide the extracted geometries as Supplementary Data. The methods could be clearer on how the segmentations for cell and DNA volume were done; were the surfaces done manually, were there any image preprocessing steps etc.? In Figure 7C, it is not clear from the images whether cells or nuclei were segmented. Also, it would strengthen the work if the authors analysed the cell shapes (in particular cell height, and apical cell shape bias), considering that they mention it to be different in the Rib mutant. In addition, it would add to the manuscript if the authors could quantify the volume of the luminal space, of the epithelial layer in wt and mutant, and the bias in tube outgrowth.
2.The authors show nicely that the rib mutants have a smaller overall cell size, can this be the reason why the secretory tube in figure is smaller? In addition, if the overall size of the mutant and the WT is the same as suggested in figure 1H then why does the mutant larvae in figure 1f appear so much smaller than the WT in the same panel?
3.In figure 4f the authors see 4 out of 7 RPGs been significantly down-regulated, do they have an explanation for that? Why are not all 7 tested RPGs significantly down-regulated? Can it be that the results will be significantly improved by dissecting the tissue of interest instead of using whole embryos? Finally with what criteria were these 7 genes selected?
4.The authors state in their manuscript the limitations of the chip-seq and the fact that the 11 unbound RPGs are essentially a technical artifact. I suggest that the authors either perform ChIP on some of these RPGs to prove their point or that they ton down their statements about chip-seq limitations and Rib binding all SG-expressed RPGs
**Minor points**
The authors need to clarify in the text what is early and late stage of tubulogenisis.
In figure 1c the Mipp1 staining is of low quality and although the white lines help the reader on where to focus, noise vs signal is almost indistinguishable. Furthermore, the authors claim that they only take under consideration SG cells that show uniform membrane staining but Figure 1c does not show such uniform staining.
Figure 1d needs the addition of statistical analysis WT vs rib mutant st12 look very similar.
In their ChIP-seq data the authors identify 436 peaks that correspond to 413 genes. It is worth to add a pie chart depicting how many of those 413 are RPGs and how may are non-ribosomal.
Throughout the manuscript the authors exhibit nicely the effects of rib mutants. What happens to the tested genes in panel 4f when rib is overexpressed?
RPls are known to be involved in size regulation. If the authors use another driver than fkh to express Rib, Rpl19 etc will they still see similar phenotypes or not?
Figure 7b is hard to follow, the IP panels should be in agreement with the order that they appear in the text e.g., first experiment then controls
Reviewer #3 (Significance (Required)):
In the manuscript "The Ribb-osome: Ribbon boosts ribosomal protein gene expression to coordinate organ form and function" the authors show evidence that Ribbon mediates early cell growth in Drosophila embryonic salivary gland through direct interaction with ribosomal protein genes. As I am only vaguely familiar with the field, I would leave it to someone who is closer to judge the advance and relevance. But with the additional quantifications, the paper should be of interest more generally to developmental biologists who are interested in tubulogenesis, and if the authors make the 3D cell geometries available, the work should also be of interest to computational modellers with an interest in epithelial organization as segmented 3D cell geometries are still rare.
**Referees cross commenting**
Looking at all 3 referee reports, I find all points made by referee 1 either essential and/or easy to fix. As such, I would insist on all points made.
With regard to referee 2, I see points 1,5,8 as essential, and point 2 is too easy to do to not request it. The others I would consider nice-to-have, but not essential.
In case of my own report, I would insist on points 1 & 2. Among the minor points, points 4 & 6 are NOT essential. The others are either important or easy enough to fix.
I look forward to the views of my colleagues.
Our response to reviewer comments
We thank the reviewers for their very positive comments regarding the importance of this paper and for the constructive feedback they have provided. Indeed, we would be delighted to address every suggestion raised, but since we would also like to have this work published in a timely manner, it is quite helpful to have consensus among the three reviewers regarding which changes and experiments are the most important to include. Since all three reviewers felt it important to address all of the comments from Reviewer #1, we will do so. For the comments raised by reviewers #2 and #3, we will follow the consensus opinion and address those comments by changes in the text or by including more experiments. In this revision plan, we also address the comments that were considered to be beyond the scope of the current study.
Points raised by Reviewer #1
Include N values for all the figure panels: We will provide sample number information for those panels currently missing that information: Figures 1F; 5B, C; and 7B.
Microarray fold-change clarification: We will clarify that we are reporting the fold-change values in Table S2. As is standard with Volcano plots for reporting microarray data, Figure 4E is plotted as Log2 data.
Antibody validation: We will provide a supplemental figure with information about the Rib antiserum and its specificity.
Add citation regarding the microarray data: We will add the citation referring to the microarray data to the Methods section.
Uncommon word usage pg 15: We will remove “viz.”—contraction of a Latin phrase “videre licet” to mean “namely” or “specifically”—from the discussion of factors in the gene network, since it was clearly distracting.
Points raised by Reviewer #2
Appearance of Nuclei and Calculation of DNA volume: The rib mutant nuclei shown in Fig. 1C depict CrebA staining and were used only for identification of SG secretory cells – we did not measure nuclear volume in these samples. To eliminate any potential confusion, we have re-labelled the last column “3D cell volume”. All of the calculations of nuclear size (as a measure of DNA amplification) were carried out with DAPI-staining as shown In Fig 1G, which revealed no difference between WT and rib mutant SG secretory cells. Measurement of entire nuclear volume is critical, since, in any single focal plane, how much of the nucleus is captured varies. We will provide information detailing how DNA volume was obtained in the methods section.
SG cell size phenotypes of M1BP and Dref RNAi Knockdowns: We agree with the reviewers that determining if M1BP and Dref SG-specific RNAi also phenocopy the cell growth deficit observed in the rib mutant SGs is a meaningful experiment and could strengthen our conclusions. We will, therefore, perform this experiment. It should be noted, however, that whereas *rib *and *Trf2 *do not have significant levels of maternal mRNA or protein, both M1BP and *Dref *have high levels of both [based on ModEncode data; Flybase]. Thus, it may be challenging to deplete these genes with only SG driven expression of the RNAi constructs.
List of potential Rib-dependent growth promoting factors in the trachea: In the revised version, we provide the list of candidate growth genes bound by Rib from the tracheal Chip-Seq data as requested by reviewer #2 (and agreed upon by reviewer #1 as important) in the supplement.
Effects of Rib overexpression on SG cell growth: All of the reviewers agree that testing for a SG secretory cell over-growth phenotype with Rib overexpression is worthwhile and we will do this experiment. Nonetheless, we recognize that we may not see overgrowth phenotypes based on a few observations. Our ChIP-Seq data indicate that Rib binds neither the promoters of ribosomal RNAs [rRNAs; the other essential component of ribosomes] nor the promoters of known rRNA transcription factors. Based on a study from another group, it seems likely that Myc upregulates rRNA expression (Grewal et al., 2005). Correspondingly, myc is transcriptionally upregulated in the embryonic SG (supplemental panel 7C) and myc expression in the SG is independent of rib (i.e. Rib does not bind the myc gene based on the SG ChIP-Seq and myc levels in the embryonic SG do not change in rib null embryos based on microarray and whole mount in situs). Also based on ChIP-Seq, Rib binds its own promoter and, based on qRT-PCR experiments, represses its own expression (Loganathan et al., 2016). Thus, over-expression of Rib with GAL4:UAS driven expression may reduce rib transcription from the endogenous locus. Nonetheless, this experiment is still worth doing.
Points raised by Reviewer #3
Information on cell segmentations: In the revised manuscript, we will provide sample 3D views of cell volume quantifications as movie files. In the methods section, we will also make it clear that the surfaces were manually segmented and that no image preprocessing steps were performed. We will also provide the excel spread sheets on size calculations in a supplement. We will provide information in the legend for figure 7 that whole secretory cells were segmented for the calculations done for panel C. The information on cell shapes, apical membrane dynamics, and luminal volumes (including the assessment of developmental dynamics of tube elongation based on live-imaging construction of computational elastic and analytical viscoelastic models) has been presented in previous publications from our lab (Cheshire et al., 2008; Loganathan et al., 2016) and from work in other labs (Blake et al., 1998). We will include this information in the revised discussion and will include the appropriate citations.
Panel 1F and comment on the apparent smaller size of the rib mutant shown: rib mutant embryos show characteristic head invagination defects along with amioserosa and dorsal closure defects [Bradley and Andrew, 2001]. The partial embryo image in Panel 1F captures the head invagination defect making the embryo appear smaller. We will include images of whole embryos in the revised version to clarify that whole embryo volumes of *rib *mutants are comparable to *WT *for the representations shown in Fig. 1F.
Clarify early vs. late Tubulogenesis: Early SGs are stage 11, 12 – when the SG cells are internalizing. Late SGs are stages 13 – 16, when the glands are fully internalized. We will clarify this in the figure legend.
Statistics on Panel 1D: We will perform statistical analysis of growth profiles shown in Fig 1D as suggested by the reviewer and include the results in the figure or figure legend.
Pie-chart for RPG fraction: Given how crowded the figures currently are, instead of providing pie charts, we simply provide the fraction of the bound genes that are RP genes in the text. Using our set cut-off of 4.0: 12.9% of genes bound by Rib (with both drivers) were RP genes. Using the IDR platform for peak calling, 12.8% of bound genes were RP genes. In Fig 4A, we also include genes above the cut-off with one GAL4 driver, but not the other, as described in the legend.
Effects of Rib Overexpression: As discussed earlier, we will perform this experiment (please also see our response to the last comment by reviewer #2)
Order of presentation of co-IP results in Panel 7B: As requested, we will reorder the IP results in Fig. 7B as suggested by the reviewer to present first the results from the experiments and then the results from controls in accord with how we discuss the data in the results section.
Testing the functional relationships between Rib and known RPG regulators: We will not determine if Rib promotes DNA binding and transcriptional activity of Trf2, M1BP, and Dref, as this experiment was considered to not be critical for this paper by any of the three reviewers.
Panel 4F and tissue-specific RT-qPCR: We agree that it would be ideal to have tissue-specific qRT-PCR, but it is not technically feasible to dissect out enough embryonic SGs for analysis (as acknowledged by Reviewer 1). In future studies, we do plan to get that kind of information from single cell RNA sequencing (scRNA-Seq) of WT and rib mutant embryos, but there are a few hurdles to overcome before those experiments. In selecting the RP genes for qRT-PCR, we chose sample RpL and RpS genes, making sure to include at least one gene (RpS9) that was “not bound” by Rib based on ChIP-Seq criteria.
Determine Rib function on RPG translation: We will not examine levels of RP proteins by Western since this experiment was deemed be unnecessary for the current study by the three reviewers.
Effects of rib on the secretory function of the SG at the embryo, larva, pupa, or adult stage: We agree with the reviewer that these data would be interesting to have; as pointed out by reviewer #1, however, it’s a question for a future follow-up study.
Chip-Seq technical artifact / limitations: We don’t think we are incorrect in suggesting that the failure to detect Rib binding to all RP genes could be a technical artifact because of the following: (1) a direct examination of the binding tracts associated with every RP gene reveals a peak at/near the TSS. The values associated with those peaks do not always reach the cut-off, but when the peak values are lower than the cut-off, the signals in the flanking DNA are often also much lower than average (for details, see Supplemental Figure 1). (2) Among the RP genes whose expression went down significantly by qRT-PCR is RpS9 – an RP gene “not bound” by Rib, based on the cut-offs we followed.
Using another SG driver: We agree with reviewer #1 that the results obtained using the fkh-GAL4 driver for RNAi of RP regulators and RP genes are robust and sufficient to support the conclusion that Rib binds RPGs to regulate SG secretory cell size. Thus, we will not redo these experiments using another SG driver.
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Referee #3
Evidence, reproducibility and clarity
In the manuscript "The Ribb-osome: Ribbon boosts ribosomal protein gene expression to coordinate organ form and function" the authors show evidence that Ribbon mediates early cell growth in Drosophila embryonic salivary gland through direct interaction with ribosomal protein genes. The manuscript is well written while presenting novel and solid data. The data could be strengthened by some further analysis and clarification, but none of the issues raised represent major flaws.
Key points:
1.Cell segmentations: The way the cell segmentations / volume quantifications are presented it is impossible to judge their quality. The authors …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #3
Evidence, reproducibility and clarity
In the manuscript "The Ribb-osome: Ribbon boosts ribosomal protein gene expression to coordinate organ form and function" the authors show evidence that Ribbon mediates early cell growth in Drosophila embryonic salivary gland through direct interaction with ribosomal protein genes. The manuscript is well written while presenting novel and solid data. The data could be strengthened by some further analysis and clarification, but none of the issues raised represent major flaws.
Key points:
1.Cell segmentations: The way the cell segmentations / volume quantifications are presented it is impossible to judge their quality. The authors should provide the extracted geometries as Supplementary Data. The methods could be clearer on how the segmentations for cell and DNA volume were done; were the surfaces done manually, were there any image preprocessing steps etc.? In Figure 7C, it is not clear from the images whether cells or nuclei were segmented. Also, it would strengthen the work if the authors analysed the cell shapes (in particular cell height, and apical cell shape bias), considering that they mention it to be different in the Rib mutant. In addition, it would add to the manuscript if the authors could quantify the volume of the luminal space, of the epithelial layer in wt and mutant, and the bias in tube outgrowth.
2.The authors show nicely that the rib mutants have a smaller overall cell size, can this be the reason why the secretory tube in figure is smaller? In addition, if the overall size of the mutant and the WT is the same as suggested in figure 1H then why does the mutant larvae in figure 1f appear so much smaller than the WT in the same panel?
3.In figure 4f the authors see 4 out of 7 RPGs been significantly down-regulated, do they have an explanation for that? Why are not all 7 tested RPGs significantly down-regulated? Can it be that the results will be significantly improved by dissecting the tissue of interest instead of using whole embryos? Finally with what criteria were these 7 genes selected?
4.The authors state in their manuscript the limitations of the chip-seq and the fact that the 11 unbound RPGs are essentially a technical artifact. I suggest that the authors either perform ChIP on some of these RPGs to prove their point or that they ton down their statements about chip-seq limitations and Rib binding all SG-expressed RPGs
Minor points
The authors need to clarify in the text what is early and late stage of tubulogenisis.
In figure 1c the Mipp1 staining is of low quality and although the white lines help the reader on where to focus, noise vs signal is almost indistinguishable. Furthermore, the authors claim that they only take under consideration SG cells that show uniform membrane staining but Figure 1c does not show such uniform staining.
Figure 1d needs the addition of statistical analysis WT vs rib mutant st12 look very similar.
In their ChIP-seq data the authors identify 436 peaks that correspond to 413 genes. It is worth to add a pie chart depicting how many of those 413 are RPGs and how may are non-ribosomal.
Throughout the manuscript the authors exhibit nicely the effects of rib mutants. What happens to the tested genes in panel 4f when rib is overexpressed?
RPls are known to be involved in size regulation. If the authors use another driver than fkh to express Rib, Rpl19 etc will they still see similar phenotypes or not?
Figure 7b is hard to follow, the IP panels should be in agreement with the order that they appear in the text e.g., first experiment then controls
Significance
In the manuscript "The Ribb-osome: Ribbon boosts ribosomal protein gene expression to coordinate organ form and function" the authors show evidence that Ribbon mediates early cell growth in Drosophila embryonic salivary gland through direct interaction with ribosomal protein genes. As I am only vaguely familiar with the field, I would leave it to someone who is closer to judge the advance and relevance. But with the additional quantifications, the paper should be of interest more generally to developmental biologists who are interested in tubulogenesis, and if the authors make the 3D cell geometries available, the work should also be of interest to computational modellers with an interest in epithelial organization as segmented 3D cell geometries are still rare.
Referees cross commenting
Looking at all 3 referee reports, I find all points made by referee 1 either essential and/or easy to fix. As such, I would insist on all points made.
With regard to referee 2, I see points 1,5,8 as essential, and point 2 is too easy to do to not request it. The others I would consider nice-to-have, but not essential.
In case of my own report, I would insist on points 1 & 2. Among the minor points, points 4 & 6 are NOT essential. The others are either important or easy enough to fix.
I look forward to the views of my colleagues.
-
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Referee #2
Evidence, reproducibility and clarity
This paper reported a role for the BTB/POZ-domain transcription factor rib in mediating early cell growth of embryonic salivary gland (SG) cells. the authors show that during tubulogenesis of the salivary glands, rib binds the transcription start site of almost all SG-expressed ribosomal protein gene (RPG) and promotes their transcription, thus providing a material foundation for cell growth. Interestingly, in embryo trachea cells, rib targets do not include RPGs, which indicates that rib may use different mechanisms to regulate cell growth of different organs. In general, this is a well-written, well designed research article with …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #2
Evidence, reproducibility and clarity
This paper reported a role for the BTB/POZ-domain transcription factor rib in mediating early cell growth of embryonic salivary gland (SG) cells. the authors show that during tubulogenesis of the salivary glands, rib binds the transcription start site of almost all SG-expressed ribosomal protein gene (RPG) and promotes their transcription, thus providing a material foundation for cell growth. Interestingly, in embryo trachea cells, rib targets do not include RPGs, which indicates that rib may use different mechanisms to regulate cell growth of different organs. In general, this is a well-written, well designed research article with many conclusions well-supported by experimental evidence. Listed below are a few issues (mostly minor/unessential) for the authors to consider.
Major comments:
1.Although in Figure 1G, the nucleus size is indistinct in rib mutant and wt cells at stage 15 and 16, Figure 1C appeared to look like that the rib mutant nuclei at stage 11, 13 and 14 are significantly smaller than those in wild type cells. The authors need to make sure that the rib phenotype has nothing to do with DNA amplification.
2.Please describe the details on calculating DNA volume by DAPI staining in the method session.
3.The authors have demonstrated weak DNA binding ability of Rib, and physical interactions between Rib with the known regulators of RPG transcription (Trf2, M1BP, and Dref), but what is the functional relationships between Rib and the known RPG regulators? e.g., does Rib function to promote DNA binding and transcriptional activity of Trf2, M1BP, and Dref, or vice versa?
4.To confirm the rib function on RPG translation, it is recommended to examine ribosomal proteins by western, and comparing the total protein content would also be helpful.
5.As Trf2, M1BP and Dref are physically interacted with Rib, it would be helpful to determine Whether M1BP and Dref knockdown can phenocopy the cell growth deficit observed in rib mutant SGs.
6.Page12, paragraph 3, "Thus, despite the shared requirement for Rib in embryonic cell growth of both tubular organs, Rib-dependent growth in the trachea is likely through regulation of alternative growth-promoting factors." Please list the potential growth-promoting factors targeted by Rib according to the Chip-seq data, if possible.
7.It would be interesting to determine whether rib mutation differently affect the secretory function of salivary gland at embryo, larva, pupa or adult stage.
8.Does Rib overexpression have any effects to SG development? Considering the authors adopted GAL4-UAS system to rescue Rib under Rib-KO, it would be interesting to see if Rib overexpression could cause an opposite overgrowth phenotype.
Significance
This paper discovered a new mechanism underlying organ-specific cell growth regulation during a specific time-window of animal development, which should be of interest to the field of cell and developmental biology.
Drosophila genetics; Developmental biology
Referees cross-commenting
I agree with all the other referees that the comments raised by reviewer #1 should be addressed entirely.
In regard to the comments by reviewer #3, all of the 4 major points are excellent and should be addressed, but it is okay to address points #3 and 4 by simple explanation or re-wording. I find the minor point #6 is nice to have but not essential, the rest should be addressed.
In case of my comments (reviewer 2), points #1,2,5,8 should be addressed, others are nice to have.
-
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Referee #1
Evidence, reproducibility and clarity
In this paper, the authors examine the relationship between the transcription factor Ribbon, its ribosomal protein gene (RPG) targets, and cell growth during the process of salivary gland tubulogenesis in the Drosophila embryo. This study builds upon previous work they published in 2016 (Loganathan et al., 2016). While the previous study identified RPGs as potential targets of Ribbon from ChIP-Seq analysis, they did not delve into the role of these targets in salivary gland morphogenesis. Here, the authors demonstrate that mutation of ribbon results in decreased cell volumes via immunostaining and image analysis. They identify and …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
In this paper, the authors examine the relationship between the transcription factor Ribbon, its ribosomal protein gene (RPG) targets, and cell growth during the process of salivary gland tubulogenesis in the Drosophila embryo. This study builds upon previous work they published in 2016 (Loganathan et al., 2016). While the previous study identified RPGs as potential targets of Ribbon from ChIP-Seq analysis, they did not delve into the role of these targets in salivary gland morphogenesis. Here, the authors demonstrate that mutation of ribbon results in decreased cell volumes via immunostaining and image analysis. They identify and confirm RPGs as ribbon transcriptional targets using ChIP-SEQ, Microarray data, in situ hybridization, and qRT-PCR. They analyze these targets in an effort to identify a Rib consensus binding sites by MEME and find that Rib binding is not specific using EMSA. They suggest specificity arises from association with transcriptional cofactors. Binding with cofactors was confirmed by CO-IP and in vivo RNAi experiments demonstrated the requirement of these cofactors in mediating changes in cell volume during salivary gland tubulogenesis. They demonstrate that Ribbon regulation of cell growth via transcription of RPGs is not a universal mechanism for Ribbon function, as Ribbon regulates transcription of other genes in the context of tracheal development.
Major comments:
Results of all experiments are conclusive, and significant numbers of samples were noted for most figure panels. For a few panels the sample number/number of replicates was not noted, and it is recommended that the authors add this information (Figure 1F; 5B,C; 7B).
Additional experiments are not needed to support the conclusions presented in this work. The data and methods are presented clearly and the statistical analyses performed were appropriate.
In regard to microarray data, Figure 4E shows fold change as log2 values, but it is unclear if this is the case for Table S2. This should be clarified. The authors note in the text on page 7 that few targets show a greater than 1.5-fold change. Based on Figure 4E, this is a log2 value, and should be specified as such.
As the Rib antibody was generated in this study, it would be helpful to include data illustrating a confirmation of antibody specificity. This could include Rib antibody staining on rib mutant embryos, or showing a lack of band for ribbon in ribbon mutants on a Western blot. If the specificity has been published elsewhere, please add a reference.
Minor Comments:
As the microarray data was previously published in Loganathan et al 2016, as mentioned in the results section, this citation should also be included in the Methods section describing the Microarray data.
In the discussion section on page 15, a list of factors in the gene network are listed. What is viz.?
Significance
•As described in the introduction, the role of cell growth during embryonic tissue morphogenesis is a relatively unexplored topic. The authors point out that most previous studies describing regulation of tissue growth have focused on the role of mitosis and increased polyploidy, as in the gut (https://doi.org/10.1016/S0925-4773(00)00512-8 ), as primary mechanisms. In the case of the salivary gland, only a single endocycle occurs during embryogenesis and cells are post-mitotic, suggesting another mechanism is at play. This study identifies Ribbon as a mediator of cell growth and demonstrates that Ribbon mediates this function through transcriptional regulation of RPGs. In addition, they identify Ribbon cofactors that are important for salivary gland cell growth and tissue morphogenesis. Interestingly, they find that this mechanism for cell growth may be tissue specific, as Ribbon appears to regulate different genes in the trachea.
•This work has implications for the regulation of cell growth in other tissues and organisms and would be of broad interest to those studying organ development.
•In order to contextualize my review, I am a developmental biologist that works with Drosophila.
Referees cross-commenting
In regard to the comments by reviewer #2: I agree that point # 2 should be addressed to more thoroughly describe the method, but as the authors have looked at DNA Amplification at a time point following the normal endocycle, which occurs at stage 12, and DNA content is not significantly different, I don't think analysis of earlier stages would influence their conclusions.
Given that the authors do include some RNAi data for RPGs and Trf2, it would enhance the paper further to include M1BP and Dref RNAi data if quality reagents are available as described in point 5. Point 6 can be easily addressed. In regard to point 8, the effects of rib overexpression alone would be interesting to see given the ability of this construct to rescue the phenotype.
While I think points 3 and 7 are excellent ideas for a follow up study, I think they are outside of the scope of this paper. I do not view point 4 as essential to this study, as the study focuses on the regulation of transcription of the RPGs by Rib.
In regard to the comments by reviewer #3, I agree that points 1 and 2 should be addressed. It would be extremely difficult to address point #3 by dissecting out the tissue, but it could be addressed via further explanation in the text, as could point #4. I don't think minor points 4-6 need to be addressed, but the minor points 1-3 should addressed to improve the paper. For minor point #3, I would suggest the number of genes be included in Supplementary Table 1.
As reviewer #1, I think my comments should be addressed to improve the quality and clarity of the paper.
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