Systematic analysis of YFP gene traps reveals common discordance between mRNA and protein across the nervous system

Article activity feed

1. 

   Review Commons

   May 27, 2022

   Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Reply to the reviewers

   Manuscript number: RC-2022-01392R

   Corresponding author(s): Ilan Davis

   General Statements

   We thank the reviewers for their constructive and helpful comments on our manuscript. We are delighted to find their consensus that the manuscript represents a useful resource for the Drosophila community in particular, and for the fields of neural development and post-transcriptional gene regulation. The following is our detailed responses and plan for how we will address all the major points raised by the reviewers. We also plan to address all minor points fully and have been through them in great detail one by one, so we are confident this is feasible within a …

   Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Reply to the reviewers

   Manuscript number: RC-2022-01392R

   Corresponding author(s): Ilan Davis

   General Statements

   We thank the reviewers for their constructive and helpful comments on our manuscript. We are delighted to find their consensus that the manuscript represents a useful resource for the Drosophila community in particular, and for the fields of neural development and post-transcriptional gene regulation. The following is our detailed responses and plan for how we will address all the major points raised by the reviewers. We also plan to address all minor points fully and have been through them in great detail one by one, so we are confident this is feasible within a reasonable and expected time frame.

   Description of the planned revisions

   Reviewer #1

   Major 1: For the wildtype CS flies, there is no YFP mRNA signal in neuroblast region and how about YFP mRNA signal in MB, OL VNC and NMJ regions? What is the criterion of setting laser power and gain for the mRNA level of 200 genes? Is it difficult to distinguish background and true signal of the mRNA in different area?

   This is a good point about background intensity levels (from non-specific binding of the YFP smFISH probe) across different tissue regions. We thank the review for raising it. Signal:background decreases with depth in all of the tissues, with superficial cells displaying similarly high signal:background in the CNS and NMJ, while signal:background in neuropil regions of the CNS are slightly lower. To address this point, we plan to include a supplementary figure to show background fluorescence of the smFISH probe across all regions of the CNS and NMJ.

   To address the point about image acquisition settings, we will included the following additional information in the Methods section (Page 17):

   “Consistent image acquisition settings (laser power, pixel dwell time or camera exposure, detector gain) were used for experimental and control experiments. Acquisition settings were optimized to achieve fast acquisition and high signal:background for each instrument.”

   We will add a further explicit explanation to the manuscript referring to previous publications, that the nature of the smFISH method makes it relatively simple to distinguish background from true signal. True punctae have a relatively uniform size, symmetrical shape, and consistent intensity distribution. Whereas background punctae that are either larger than diffraction-limited punctae or have lower intensity can easily be separated from real signal.

   Major 2: Would the insertion of YFP affect gene expression? Comparing to CS in Fig 1K, the dlg1 mRNA signals in dlg1::YFP line (Fig 1F) increases a lot. I do not know if this phenotype happens only in this area. So could you show some other regions for dlg1::YFP flies.

   This is a good point raised by both Reviewer #1 and Reviewer #2 (Major point 1). We agree that a proper quantification of the effect of YFP-insertion will bolster our conclusion, highlighting the utility of protein-trap collections for systematic analysis of post-transcriptional regulations. To address this, we plan to: (i) provide quantifications of dlg1 transcript expression in the CNS and NMJ and compare the levels between dlg1::YFP and wild-type lines, and (ii) provide new figure visuals reflecting our quantification results.

   Major 3: Is the dlg::YFP homozygous available? Among 200 gene trap lines, how many of them can be homozygous?

   This is a good point raised by both Reviewer #1 and Reviewer #3 (Major point 1). The dlg1::YFP (CPTI-000207) line used for the control experiments is homozygous. However, it is a great point that not all of the YFP insertions are homozygous viable. Out of the 200 lines we screened, 131/200 (65.5%) insertions are homozygous viable, whereas 69/200 (34.5%) are homozygous lethal or are unknown. We have addressed this caveat in the Methods section (Page 16) with the following statement:

   “The majority of YFP insertion lines are homozygous (65.5%, 131/200), those that are not homozygous viable were kept over balancer chromosomes.”

   Our provisional analysis shows that the number of nervous system compartments expressing YFP-fused protein or mRNA are not affected by homozygous lethality. We plan to include this analysis in the revised manuscript.

   Major 4: Have you tried to investigate the mRNA and protein localization in adult brains?

   Yes, in a related study, we demonstrated that this approach also works in the adult brain (Mitchel et al., 2021, DOI:10.7554/eLife.62770). A systematic analysis of protein and mRNA expression patterns in the adult brain would be highly interesting and is certainly possible, however it is beyond the scope of the manuscript. To address this point, we will cite our related work and emphasise more clearly the wider applicability of our technique.

   Major 5: In Fig 3C, the authors claimed in MB or OL soma regions, some genes are protein expression only but no mRNA present. I wonder how do you explain this phenotype in soma.

   Our favoured explanation is that protein is more stable than mRNA. Therefore, after the mRNA is translated, it could get degraded while the protein is still present in the cell. We will add text in the relevant section to mention potential differential stability of protein/mRNA.

   __Major 6: Since sgg mRNA localize to both sides of NMJ, would KCl stimulus affect sgg mRNA amount and localization in muscle? __

   That is an interesting question. The data in Fig. 8I-J show that there is no additional Sgg::YFP protein accumulation at the muscle post synaptic density in response to KCl stimulus. It’s been shown elsewhere (Ataman et al., 2008, DOI:10.1016/j.neuron.2008.01.026) that Sgg protein translocates to the muscle nucleus in response to KCl stimulus. Determining whether that mechanism requires translation of new protein would require a complete new study with translational analysis and would distract from the message of the current study.

   Reviewer #2

   Major 1: Although the group is using an established and published set of gene traps, it would be good to confirm protein expression for same gene to increase confidence or provide more details on how is known that the YFP insertions do not affect mRNA stabilization or transcription or protein expression/localization. For example in Figure 1 F' versus K it is unclear why in the DlgYFP insertion there are more Dlg in situ signals than are observed in and around a neuroblast as compared to the wild type control. From the description provided these appear to the maximum intensity images. Is this due to background or an effect of the YFP insertion itself? Because of the increased level of expression is there a feedback loop of the protein regulating the mRNA expression? If had expression of Dlg protein in this figure would also confirm the YFP insertion mirrored the endogenous and it would be easier to discern if there were any changes in the number of Dlg mRNA molecules present. As this was the proof of principle example for the screen this information would increase confidence in the remainder of the data presented. AS an important part of the screen is looking at the potential for post transcriptional regulation this is an important factor to address.

   Thank you for the valuable suggestion. We agree with the reviewer that the comparison of dlg1 transcript levels would provide a valuable control. This point was raised by both Reviewer #1 and Reviewer #2. Please see [Reviewer #1 - Major point 2] for our response.

   __Major 2: Will this pipeline capture information on whether is secreted (contain a signal regulatory peptide) or not as then would expect to be discordant. This should be clarified or commented on. __

   The reviewer’s comment is correct. Secreted proteins may show discordant distribution of protein and mRNA between cell types even in absence of post-transcriptional regulations. Note that Shaggy (Sgg) is a secreted protein but we observe that most of the protein products are expressed in the same cell as the RNA. We propose to follow the reviewer’s suggestion and revise the text to discuss the limitation of our pipeline in identifying proteins regulated via secretory modes.

   Major 3: General molecular function is listed in supplementary table 1 but will other types of information be able to be correlated from datasets or databases as well.

   This question highlights a major feature of our dataset and associated metadata The analysis in Supplementary Table 1 is used to assess the functional representation of the 200 genes in our screen against the all known genes. We found that ~90% of GOSlim terms are covered by the 200 genes, highlighting the diversity of our list of genes. On the other hand, our Zegami resource (Accompanying data for Zegami) contains a rich collection of metadata (including the full list of GO terms) associated with each gene in the dataset, and extends that information to the entire genome. We anticipate that the Zegami resource will be a valuable platform to query data from our analysis and other databases. To address this, we plan to: (i) revise the legend for the Supplementary Table 1, and (ii) revise the text to clarify what kind of information is available in our Zegami resource.

   Reviewer #3

   Major 1: The approach relies on gene traps that often fail to be made homozygous, presumably due to deleterious function of the YFP insert. This is an obvious limitation of the study, which the authors address, but do so insufficiently by only analyzing a single case Dlg1. The authors should report how many of the 200 YFP-traps can produce viable homozygous animals, whether phenotypes can be observed, and any other relevant information to assess the functional properties of the tagged genes.

   Thank you for requesting further information on homozygous viability of the YFP-trap collection. This point was raised by both Reviewer #1 and Reviewer #3. Please see [Reviewer #1 - Major point 3] for our response.

   Major 2: The term "discordant" is used for non-congruous RNA/Protein levels in soma and distal processes, and sometimes the two are analyzed in the same figure (e.g Fig 3A). When it is stated that 98% of genes are discordant, this is an over-simplification as what the authors describe as "discordant" is expected to occur frequently in the distal process, but less often in the soma (which is what the authors find when presenting the data for individual compartments - Fig 3B-C). This is confusing because the observation means completely different things in the two compartments, though both are interesting to describe. These analyses, and their interpretation, should be kept separate.

   This is a fair point raised by the reviewer. To address this point we plan to: (i) prepare two separate tables summarising our annotation in soma and neurite compartments, and (ii) revise the text accordingly to explain and discuss how the discordant protein and mRNA expression pattern can arise both within different compartments of a cell or between different cell types in a cell lineage

   Major 3: There is not enough emphasis placed on the cell-type specific regulation of RNAs. There are very few studies that have investigated how localization of individual RNAs changes in different cell types or regions of the nervous system, and the authors find that this is quite prevalent. Therefore, the rather superficial analysis of these data fails to take advantage of a major strength of the data. For example, for the discordant genes that differ in neuropil localization between different regions of the CNS, what types of molecules do they encode, what is their function in neurons (if known), and why might they be required locally in one region of the CNS but not the other?

   We appreciate that the Reviewer recognizes the power of comparing RNA localization patterns across different brain regions (Figure 5R). We reported on a common set of synaptic mRNAs that encode nuclear proteins across the different regions of the nervous system. Per the Reviewer’s suggestion, we have begun to look into region-specific patterns of expression. In Figure 5R, two categories with the largest number of genes are ‘protein_MB_syn’ and ‘protein_OL_syn’, which contain proteins that are specific to those regions. However, given the small number of 15-16 genes, gene ontology enrichment analysis has limited power to infer information on the entire genome.

   We plan to revise the manuscript:

   to include tables with lists of genes specific to MB and OL regions. to revise the manuscript to include in the discussion a caveat of the limited power of analysis based on a small number of genes.

   Major 4: The authors conclude that mRNA and protein co-localization in glia processes shows that mRNA localization makes a major contribution of the proteome in processes. However, there is not enough evidence for such conclusion since neither translation of these mRNAs nor lack of protein trafficking from the somas was shown.

   The significant role of RNA localisation in shaping the local proteome and performing proteostatic regulation has been studied in detail (Zappulo et al., 2017, von Kugelgen and Chekulaeva 2022 Giandomenico et al., 2022). However, the reviewer’s comment is correct that we do not show direct evidence of mRNA translation or protein trafficking. Therefore, we propose to: (i) clarify the text by including the citation of these publications, and (ii) qualify our claim that mRNA localization is a major contribution of the proteome in neurite or glial processes.

   Zappulo et al., 2017, DOI: 10.1038/s41467-017-00690-6

   von Kugelgen and Chekulaeva 2022 DOI: 10.1002/wrna.1590

   Giandomenico et al., 2022, DOI: 10.1016/j.tins.2021.08.002

   Major 5: An important caveat of this technique that should be discussed is the lack of knowledge about the translation of these mRNAs, if the mRNA that is being detected is the same as the one that is translated. While the authors emphasize the discordance between mRNA and protein localization, it is not possible to know whether these mRNAs are being translated where they are found, e.g. soma vs neuropil. Moreover, there are many examples (e.g. BDNF) where the isoform influences the subcellular localization of the mRNA. There is no way of studying the isoforms here, and we could be looking for a different mRNA isoform localized to a specific compartment compared to the protein. These points must be discussed.

   We agree with the reviewer that our method does not provide information on whether the detected mRNA is being translated in time and space. Elucidating the relative contribution of localised mRNA in shaping the local proteome is not a trivial task and it is being actively investigated in the field. However, we believe our dataset provides a unique high-resolution map of transcripts that are potentially regulated at post-transcriptional and translational levels. It would be promising to follow up the ‘discordant’ genes identified from our survey using experimental methods that are able to track mRNA-ribosome associations (e.g. TRICK) in future studies. To address this point, we will revise the text to discuss this caveat.

   Thank you for pointing out the matter with mRNA isoforms. Our preliminary analysis indicates that 71% of the screened genes have constitutive YFP-insertions (i.e. YFP-cassette traps all mRNA isoforms). However, we agree that our approach cannot discriminate the case where protein produced from an mRNA isoform is trafficked and co-localises with another mRNA isoform that did not give rise to that protein. We plan to revise the text to discuss this point explicitly.

   Description of the revisions that have already been incorporated in the transferred manuscript

   Several minor comments regarding typos and simple errors have already been incorporated in the transferred manuscript. The changes are highlighted in yellow in the revised submission.

   We plan to address all the useful numerous minor comments that the reviewers have kindly highlighted to us. We feel these are straightforward to do and feasible in a short time, so do not require a detailed listed plan. If the reviewers feel they do afterall need such a list, we will be happy to provide it. However, there is one minor comment that we feel requires a little more explanation:

   Description of analyses that authors prefer not to carry out

   Reviewer #3 - Minor Comment on Figure 8: “...____*they should characterize the (khc) mutant NMJs: what is the change in size, synapse number, etc.. *

   The khc mutants are already known to show synapse morphology phenotypes (Kang et al., 2014), though the khc23/khc27 transheterozygous allele has previously been used to assess localization defects at the larval NMJ (Gardiol and St. Johnston, 2014). Moreover, our manuscript (Figure 8) focuses on post-developmental stimulus-dependent processes, rather than cellular-level synapse developmental parameters with this mutant. The reviewer correctly points out that the khc developmental phenotypes are likely to have other secondary defects as a result of impaired microtubule transport. The purpose of that mutant was to assess the molecular-level question of whether microtubule-based transport is required for sgg mRNA localization at the axon terminal. The consequences and exact mechanism of disrupted transport are beyond the scope of this study. To address this point explicitly, we will:

   Revise the manuscript to quote more explicitly and clearly the developmental khc phenotype. Revise the manuscript to explain the difference between the developmental role of khc and role in the transport of sgg specifically to the axon terminal. Revise the manuscript to explain more explicitly the limitations of this mutant.

   Read the original source
2. 

   Review Commons

   May 27, 2022

   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 this manuscript, the authors address the important topic of post-transcriptional gene regulation using the larval nervous system in Drosophila. They utilize a novel approach taking advantage of existing protein trap library, which permits use of the same smFISH probe to detect an array of 200 RNAs and visualize their corresponding protein expression. Furthermore, the authors developed a computational pipeline to visualize and analyze the resulting data, which should enhance the application of this method by other researchers. A major strength of the data comes from the analysis of multiple cell types in distinct compartments of …

   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 this manuscript, the authors address the important topic of post-transcriptional gene regulation using the larval nervous system in Drosophila. They utilize a novel approach taking advantage of existing protein trap library, which permits use of the same smFISH probe to detect an array of 200 RNAs and visualize their corresponding protein expression. Furthermore, the authors developed a computational pipeline to visualize and analyze the resulting data, which should enhance the application of this method by other researchers. A major strength of the data comes from the analysis of multiple cell types in distinct compartments of the nervous system, cell types (neuron, glia, neuroblast), and subcellular domains. From the cumulative data, the authors are able to describe several interesting observations relating to cell-specific post-transcriptional regulation, regulation within a central-neuroblast lineage and glial post-transcriptional regulation, among others.

   However, in spite of these strengths, there are several concerns related to the organization and interpretation of the manuscript that the authors should address in order to improve the manuscript:

   General concerns:

   1. The approach relies on gene traps that often fail to be made homozygous, presumably due to deleterious function of the YFP insert. This is an obvious limitation of the study, which the authors address, but do so insufficiently by only analyzing a single case Dlg1. The authors should report how many of the 200 YFP-traps can produce viable homozygous animals, whether phenotypes can be observed, and any other relevant information to assess the functional properties of the tagged genes.
   2. The term "discordant" is used for non-congruous RNA/Protein levels in soma and distal processes, and sometimes the two are analyzed in the same figure (e.g Fig 3A). When it is stated that 98% of genes are discordant, this is an over-simplification as what the authors describe as "discordant" is expected to occur frequently in the distal process, but less often in the soma (which is what the authors find when presenting the data for individual compartments - Fig 3B-C). This is confusing because the observation means completely different things in the two compartments, though both are interesting to describe. These analyses, and their interpretation, should be kept separate.
   3. There is not enough emphasis placed on the cell-type specific regulation of RNAs. There are very few studies that have investigated how localization of individual RNAs changes in different cell types or regions of the nervous system, and the authors find that this is quite prevalent. Therefore, the rather superficial analysis of these data fails to take advantage of a major strength of the data. For example, for the discordant genes that differ in neuropil localization between different regions of the CNS, what types of molecules do they encode, what is their function in neurons (if known), and why might they be required locally in one region of the CNS but not the other?
   4. The authors conclude that mRNA and protein co-localization in glia processes shows that mRNA localization makes a major contribution of the proteome in processes. However, there is not enough evidence for such conclusion since neither translation of these mRNAs nor lack of protein trafficking from the somas was shown.
   5. An important caveat of this technique that should be discussed is the lack of knowledge about the translation of these mRNAs, if the mRNA that is being detected is the same as the one that is translated. While the authors emphasize the discordance between mRNA and protein localization, it is not possible to know whether these mRNAs are being translated where they are found, e.g. soma vs neuropil. Moreover, there are many examples (e.g. BDNF) where the isoform influences the subcellular localization of the mRNA. There is no way of studying the isoforms here, and we could be looking for a different mRNA isoform localized to a specific compartment compared to the protein. These points must be discussed.

   Minor suggestions:

   • The authors should identify GO terms to understand what types of molecules are subjected to RNA regulation. They provide a supplementary table for all genes, but it would be useful to have a chart showing the proportion of different GO terms represented in the overall gene set, genes that show cell-specific regulation, genes that show neuron vs glia specific regulation, etc.
   • "However, post-transcriptional regulation can also manifest itself within a cell, so that a protein is localised to a distinct site from the mRNA that encodes it". While subcellular RNA localization may represent a regulatory layer, I do not agree that proteins that function in the cell at a different location than their translation site represents regulation per se. Many such cases exist for proteins that are trafficked!
   • "The majority of individual puncta appearing in the dlg1::YFP line (51% in the brain, 64% in larval muscles". Why is the agreement between YFP and endogenous FISH so low? Do many individual RNAs fail to hybridize? This should be discussed.
   • "However, one gene, indy, is highly transcribed in neuroblasts and a single ganglion mother cell before it is rapidly shut off (Figure S1A)". This figure does not exist. Where are the data?
   • The authors should be consistent about calling perineurial or perineural glia (both correct) in their images and text.
   • "We only observe a minority of localised axonal mRNAs that lack the protein they encode at the axon extremities, in contrast to our findings in the mushroom body, optic lobe, and ventral nerve cord neuropils" These results are not contrasted, as in all neuropils the minority of localized mRNAs are those lacking their corresponding proteins. For example, 9% in NMJ vs 7.5% in OL neuropil according to Fig. 1B. What is conflicting with the conclusion?
   • "These results suggest that motor axons are more selective than the other neuronal extensions in the mRNAs that are transported over their very long distances from the soma to the neuromuscular synapse" The current literature says that the same mechanism (cis-elements) is used to transport mRNAs to subcellular compartments, which would be inconsistent with the idea of motor axons being "more selective" than other neurons for the same mRNA, but just a result of fewer mRNAs being found in motor neurons: 34.% of the mRNAs are found in motor neurons soma vs 83% in OL soma, 86.5% in VNC soma, and 70.5% in MB soma. To get to this conclusion, the authors should show that mRNAs previously found in the neuronal extensions of other neurons are not found in the axons of motor neurons but are still expressed in thesir somas. They might want to suggest different RBPs involved in the transport or discussing the very long distance they need to travel which can influence their detection in the tips. Figures
   • Figure 1. Experimental approach summary
     • Some colors do not show well and should be changed, e.g: grey in Fig. 1A, and Fig. 1B probe sites indicated in light blue and pink within the introns of dlg1.
     • Fig. 1E': There appears to be a large discrepancy in co-detection % for CNS and muscle in the graph judging by the size of circles, yet in the text, it is stated that there is average of 51% and 64% in the two, respectively. I don't see any green circles with over 25% agreement in the graph. Are the colors correct here?
     • Fig. 1D-I: It's difficult to identify where the zoomed panels come from. E has its own square (indicating zoom in E'). Please make this square dashed or a different color in E so it is clear F and G do not come from there.
     • Comparing Fig. 1F vs K: Why does there appear to be so much more dlg1 mRNA in the YFP-tag condition? If this is due to selection of imaging area, please choose a more similar region to image so the RNA levels are comparable. Otherwise it indicates the YFP-tag line has more RNA expression, which is likely not the case.
   • Figure 2. Analysis pipeline overview
     • The lines for the first two zoomed panels are switched: The optic lobe is going to VNC and vice-versa.
   • Figure 3. Overall summary of results
     • Figure 3A: Soma/Neuropil/muscle should be separate or at least ordered such that they are next to each other to facilitate direct comparison of genes in the same region of the cell in neurons from different CNS areas. Why are glia not included in this summary? A third color should be used to indicate when there is neither mRNA nor protein expression.
     • "Compiling all the information together shows that there are that 196/200 or 98% of the genes show discordance between RNA and protein expression" However, 5 genes shown in Fig. 3A do not show "discordance": CG9650, cup, Lasb, rg, and vsg!!
   • Figure 4. Neuroblast lineage analysis
     • Is clustering around the NB sufficient to determine lineage relationship? There seems to be other neurons around the NB.
     • More examples should be shown for the post-transcriptional category, as it is the most interesting category, and there are many different possible outcomes. Are there cases of transcriptional control and post-transcriptional regulation? Are there cases where the youngest neurons (closer to the NB) in the progeny are expressing the protein while the oldest are not? If not, could this be an artifact from a slow translation and the protein being detected only after building up in the cell? Top1 protein (Fig. 4D) seems to be less expressed in the youngest neurons.
     • "The transcription rate of these genes, as indicated by the relative intensity of smFISH nuclear transcription foci, is similar across the neuroblast lineage, however protein signal is only detectable in a minority of the progeny cells (Figure 4E)". Many nuclei lack clear large spots, but have small spots indicative of RNA; how is this interpreted? Do they lack transcription, or is this due failure of the smFISH to capture all transcription sites? Were transcripts actually counted to assess cell-specific differences? This should be possible with smFISH
   • Figure 5. RNA synaptic localization
     • A have global analysis comparison of all neuropil areas would be welcome in this figure.
     • "Surprisingly, another 59 transcripts are present at synapses without detectable levels of protein (Figure 5E-H)" This text does not correspond to Fig 5E-H but 5I-L. Where is the text about 5E-H?
     • For Fig. 5J and 5N RNA appears scattered regularly throughout the entire panel area. How sure are the authors that this is not due to poor signal/noise? For example, perhaps too much probe being used for these targets.
     • Fig. 5R is not cited in the text.
   • Figure 6. RNA localization in glia
     • For Fig. 6B-G it is hard to tell if there is any overlap of the RNA and Glia. Maybe show multiple zoomed-in merged images and/or highlight the structures with lines that are present in all panels.
     • For Fig. 6L-O: How reproducible is this small amount of RNA puncta in the NMJ glia? Is this possibly biologically important?
     • Why do cartoons labelling subnuclear/perinuclear glia in Fig.6 and Fig.S6 show different localization?
     • The cartoons seem to extrapolate from the data: While in Fig 6B-D, we see neither the big bright spot of transcription in the glial nucleus nor as many transcripts in the neuropil, they are both present in the cartoon. In Fig. 6E-G there is no indication of cortical glia soma nor the transcription spot only in glia nuclei.
     • "To assess glial localisation for the 200 genes of interest, we used a pan-glial gal4 driving a membrane mCherry marker (repo-GAL4>UAS-mcd8-mCherry) to learn the expression pattern of all glial cells, and then classified the pattern in the YFP lines (without the marker) based on knowledge of that expression pattern. We validated this approach by combining the RFP marker" Did the authors use mCherry or RFP for these experiments? Also, the previous sentence is redundant.
   • Figure 7. RNA localization at neuromuscular synapse
     • RNA for these genes seems far too spread throughout the muscle to draw any conclusions
     • Also with so many RNAs distributed in the muscle, specific localization of RNA molecule to the precise PSD would have no conceivable benefit
     • I suggest drawing lines around the protein expression to facilitate visualization of the mRNA localization for panels B, F and J. It is especially hard to conclude anything from panels B and F.
     • Light grey with white dots is hard to see in the cartoons
   • Figure 8. Role of khc and activity in sgg localization
     • Presumably there is a huge number of developmental problems associated with this mutant that could cause decrease in sgg localization
     • If the authors include this, then they should characterize the mutant NMJs: what is the change in size, synapse number, etc..
     • Is there more sgg accumulated in soma as a result of less transport? Is sgg being expressed at the same level?
     • Fig. 8F-H: Why is Dlg1 accumulated in the entire axon, not just the presume synapse?
     • Fig. 8J: Why is sgg signal occurring in circles disconnected from the main axon? The authors should show a different image

   Significance

   This is a significant and complex paper that contributes with novel tools to an important issue

   Read the original source
3. 

   Review Commons

   May 27, 2022

   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

   Summary

   Titlow et al present a data resource paper for mRNA localization and protein expression in vivo focusing on the larval nervous system which is an area of high interest currently. They screen a known group of YFP gene trap lines (200 lines) and looked at specific aspects of the nervous system such as expression in neuroblasts, the mushroom bodies, glia or the NMJ. They also present a computational workflow using this set of 200 genes for the investigation of the subcellular localization and potential role of post transcriptional regulation in whole larval tissues. This uses the image data obtained experimentally and then …

   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

   Summary

   Titlow et al present a data resource paper for mRNA localization and protein expression in vivo focusing on the larval nervous system which is an area of high interest currently. They screen a known group of YFP gene trap lines (200 lines) and looked at specific aspects of the nervous system such as expression in neuroblasts, the mushroom bodies, glia or the NMJ. They also present a computational workflow using this set of 200 genes for the investigation of the subcellular localization and potential role of post transcriptional regulation in whole larval tissues. This uses the image data obtained experimentally and then compares with existing datasets to obtain more information.

   Major comments

   The authors results largely support the claims made in the manuscript. Is a clear proof of concept analysis of specific examples and then presentation of examples from different part of the nervous system. Different aspects of the gene trap lines are taken into account. Is a high level analysis of the sub cellular localization of mRNA and protein in different parts of the nervous system. Some interesting new insights which can lead to more in depth analysis of mechanism are presented. Is an interesting idea and presents a method in which to approach a fieId that has many remaining open questions. This manuscript is an important and timely analysis that will be of high interest in the field.
   Is a positive that the authors confirmed the YFP mRNA in situs with an endogenous gene in situ. Although the group is using an established and published set of gene traps, it would be good to confirm protein expression for same gene to increase confidence or provide more details on how is known that the YFP insertions do not affect mRNA stabilization or transcription or protein expression/localization. For example in Figure 1 F' versus K it is unclear why in the DlgYFP insertion there are more Dlg in situ signals than are observed in and around a neuroblast as compared to the wild type control. From the description provided these appear to the maximum intensity images. Is this due to background or an effect of the YFP insertion itself? Because of the increased level of expression is there a feedback loop of the protein regulating the mRNA expression? If had expression of Dlg protein in this figure would also confirm the YFP insertion mirrored the endogenous and it would be easier to discern if there were any changes in the number of Dlg mRNA molecules present. As this was the proof of principle example for the screen this information would increase confidence in the remainder of the data presented. AS an important part of the screen is looking at the potential for post transcriptional regulation this is an important factor to address Will this pipeline capture information on whether is secreted (contain a signal regulatory peptide) or not as then would expect to be discordant. This should be clarified or commented on. General molecular function is listed in supplementary table 1 but will other types of information be able to be correlated from datasets or databases as well.

   Minor comments

   On page 9 refer to Figure 6S which I think is supposed to be Figure S6. In text refer to an example of gli but show gs2 in the figure so it is unclear what is being referred to or shown. Could include more description on the generation of the supplementary tables and analysis of the tables. I could not find any description/legend which made analysis of some of the tables more difficult. The data set was trained on a known set of data (analyzed by experts. It would be interesting to see what it could do with a novel set of genes in the context of post transcriptional regulation, but that is beyond the overall scope of this manuscript.

   Significance

   This is an interesting idea and is a useful resource for the genes analyzed. Gives an initial tool to analyze the expression of genes. Allows for systematic analysis of mRNA (smFISH) and protein on a larger scale but with high resolution. Adds new knowledge in terms of the localization of mRNAs and protein in the periphery of neural and glia processes which may inform future analyses of the role of these genes in these tissues.

   Is a useful resource within neurodevelopment in Drosophila and post transcriptional regulation. Would be of interest to a general audience as workflow could be applied to any tissue or set of genes. Covers a very broad set of genes with disparate biological functions again making this of interest to a broader audience.

   Expertise of reviewer Drosophila, neurodevelopment, RNA regulation, post transcriptional regulation, polarity and adhesion.

   Read the original source
4. 

   Review Commons

   May 27, 2022

   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

   This manuscript by Titlow et al. systematically analyzed spatial distribution of 200 gene's mRNA and protein, and found common discordance between them. Moreover, the browsable resource is pretty useful to most fly people. Though the authors did huge amount of experiments and analysis, and got several really interesting findings, there are some basic questions need to be answered.

   Major 1: For the wildtype CS flies, there is no YFP mRNA signal in neuroblast region and how about YFP mRNA signal in MB, OL VNC and NMJ regions? What is the criterion of setting laser power and gain for the mRNA level of 200 genes? Is it difficult to …

   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

   This manuscript by Titlow et al. systematically analyzed spatial distribution of 200 gene's mRNA and protein, and found common discordance between them. Moreover, the browsable resource is pretty useful to most fly people. Though the authors did huge amount of experiments and analysis, and got several really interesting findings, there are some basic questions need to be answered.

   Major 1: For the wildtype CS flies, there is no YFP mRNA signal in neuroblast region and how about YFP mRNA signal in MB, OL VNC and NMJ regions? What is the criterion of setting laser power and gain for the mRNA level of 200 genes? Is it difficult to distinguish background and true signal of the mRNA in different area?

   Major 2: Would the insertion of YFP affect gene expression? Comparing to CS in Fig 1K, the dlg1 mRNA signals in dlg1::YFP line (Fig 1F) increases a lot. I do not know if this phenotype happens only in this area. So could you show some other regions for dlg1::YFP flies.

   Major 3: Is the dlg::YFP homozygous available? Among 200 gene trap lines, how many of them can be homozygous?

   Major 4: Have you tried to investigate the mRNA and protein localization in adult brains?

   Major 5: In Fig 3C, the authors claimed in MB or OL soma regions, some genes are protein expression only but no mRNA present. I wonder how do you explain this phenotype in soma.

   Major 6: Since sgg mRNA localize to both sides of NMJ, would KCl stimulus affect sgg mRNA amount and localization in muscle?

   Minor 1: You claimed that Fig 1E shows high magnification image of the inset in D, but the scale bars are the same.

   Minor 2: Figure 1 legend: K-N, are the images individual channels shown in E? Or in J?

   Minor 3: In Fig 2A, optic lobe neuropil and VNC neuropil are mislabeled.

   Minor 4: Only one panel has scale bar in Fig 4.

   Minor 5: What is Fig 5B'and F'? You should describe them in the Figure legends.

   Significance

   The browsable resource is pretty useful to most fly people. The authors did huge amount of experiments and analysis, and got several really interesting and important findings.This work will provide mRNA localization information for post-transcriptional regulation studies.

   Read the original source
5. 

   Version published to 10.1101/2022.03.21.485142v2 on bioRxiv

   Mar 24, 2022
6. 

   Version published to 10.1101/2022.03.21.485142v1 on bioRxiv

   Mar 21, 2022