Mammalian glial protrusion transcriptomes predict localization of Drosophila glial transcripts required for synaptic plasticity

  1. Dalia S. Gala
  2. Jeffrey Y. Lee
  3. Maria Kiourlappou
  4. Joshua S. Titlow
  5. Rita O. Teodoro
  6. Ilan Davis

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Abstract

The polarization of cells often involves the transport of specific mRNAs and their localized translation in distal projections. Neurons and glia both contain long cytoplasmic processes with important functions. mRNA localization has been studied extensively in neurons, but very little in glia, especially in intact nervous systems. Here, we predicted 1,700 localized Drosophila glial transcripts by extrapolating from our meta-analysis of 8 existing studies characterizing the localized transcriptomes and translatomes of synaptically-associated mammalian glia. We tested these predictions in glia of the neuromuscular junction of Drosophila larvae and found that localization to mammalian glia is a strong predictor of mRNA localization of the high confidence Drosophila homologues. We further showed that some of these localized transcripts are required in glia for plasticity of the neuromuscular junction synapses. We conclude that peripheral glial mRNA localization is a common and conserved phenomenon and propose that it is likely to be functionally important.

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    Reply to the reviewers

    Manuscript number: RC-2023-01938R

    Corresponding author(s): Ilan, Davis

    1. General Statements

    We thank all four reviewers for their helpful and constructive comments. We have gone through each and every comment and proposed how we would address each point raised by the reviewers. We are confident our proposed revisions are feasible within a reasonable and expected time frame. Some of the comments regarding minor typo/aesthetics and extra references have already been addressed in the transferred manuscript. The changes are highlighted in yellow in the transferred manuscript.

    2. Description of the planned revisions

    Reviewer #1 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

    Major points:

    1. * The presented work itself (Figures 1-4) does not need significant adjustments prior to publication, in my view, with only a few points to address. However, the work in Figure 5- doesn't really support the claims the authors make on its own, and would require some additional experiments or at the very least discussion of the caveats to its current form. * * *

    We thank the reviewer for these comments and will follow the reviewer’s suggestion by discussing the caveats regarding the interpretation of Figure 5. We will also add to the discussion to suggest future research approaches beyond the scope of this manuscript that would address the functional importance of localised mRNA translation. We will briefly mention in the discussion methods such as the quantification of the mRNA foci and the disruption of the mRNA localisation signals to disrupt localised translation and the use of techniques such as Sun-Tag (Tanenbaum et al, 2014) and FLARIM (Richer et al, 2021) to visualise local translation directly.

    Tanenbaum et al, 2014 DOI: 10.1016/j.cell.2014.09.039

    Richer et al, 2021 DOI: 10.1101/2021.08.13.456301

    __ Localized glia transcripts, are they "glial/CNS/PNS" significant or are they similar to other known datasets of protrusion transcriptomes? The authors compared their 4801 "total" localized to a local transcriptome dataset from the Chekulaeva lab finding that a significant fraction are localized in both. As the authors note, this is in good agreement with a recent paper from the Talifarro lab showing conservation of localization of mRNAs across different cell types. What the authors haven't done here, is further test this by looking at other non-neuronal projection transcriptomic datasets (for example Mardakheh Developmental Cell 2015, among others). If the predicted glia-localized processes are similar to non-neuronal processes transcriptomes, this would further strengthen this claim and rule out some level of CNS/PNS derived linage driving the similarities between glia and neuronal localized transcripts. __*

    * *

    This is a good point and we thank the review for pointing out this interesting cancer data set. We will do as the reviewer suggests and intersect our data with Mardakheh Dev Cell 2015 to test the further generality of localisation in neurons and glia, in other cell types. Specifically, we plan to intersect both glial (this study) and neuronal (von Kuegelgen & Chekulaeva, 2020) dataset with protrusive breast cancer cells (Mardakeh et al, 2015).

    von Kuegelgen & Chekulaeva, 2020 DOI: 10.1002/wrna.1590

    Mardakeh et al, 2015 DOI: 10.1016/j.devcel.2015.10.005

    __ The presentation/discussion around Figure 3 is a bit weaker than other parts of the manuscript, and it doesn't really contribute to the story in its current form. Notably there is no discussion about the significance of glia in neurological disorders until the very end of the manuscript (page 21), meaning when its first brought up.. it just sits there as a one off side point. The authors might consider strengthening/tightening up the discussion here, if they really want to keep it as a solo main figure rather than integrating it somewhere else/putting it into supplemental. In my view, Figures 2 & 3 should be merged into something a bit more streamlined. __*

    This is a good point. We plan to strengthen the presentation of Figure 3 and discussion of the significance of glia in neurological disorders by adding a description of the Figure in the Results section and highlighting the significance of glia in nervous system disorders in the Discussion section.

    __ Why aren't there more examples of different mRNAs in Figure 4? Seems a waste to kick them all to supplemental. __*

    We agree that it could be helpful to show different expression patterns in the main figure. To address this point we will add Pdi (Fig. S4D), which shows mRNA expression in both the glia and the surrounding muscle cell. This pattern is in contrast to Gs2, which is highly specific to glial cells. We will also note that although pdi mRNA is present in both the glia and muscle, Pdi protein is only abundant in the glia, suggesting that translation of pdi mRNA to protein is regulated in a cell-specific manner.

    * The plasticity experiments, while creative, I think need to be approached far more cautiously in their interpretation. Given that the siRNAs will completely deplete these mRNAs- it really needs to be stressed any/all of the effects seen could just be the result of "defective" or "altered" states in this glial population- which has spill over effects on plasticity in at the NMJ. Without directly visualizing if these mRNAs are locally translated in these processes and assessing if their translation is modulated by their plasticity paradigm, all these experiments can say is that these RNAs are needed in glia to modulate ghost bouton formation in axons. This represents the weakest part of this manuscript, and the part that I feel does not actually backup the claims currently being made. Without any experiments to A. quantify how much of these transcripts are localized vs in the cell body of these glia, B. visualize/quantify the translation of these mRNAs during baseline and during plasticity; the authors cannot use these data to claim that localized mRNAs are required for synaptic plasticity. *

    We are grateful to the reviewer for pointing out that we were not precise enough in defining our interpretation of the structural plasticity assay. We did not intend to claim that our results show that local translation of these transcripts is necessary for plasticity, only that these transcripts are localized and are required in the glia for plasticity in the adjacent neuron (in which the transcript levels are not disrupted in the experiment). Definitively proving that these transcripts are required locally and translated in response to synaptic activity would require genetic/chemical perturbations and imaging assays that would require a year or more to complete, so are beyond the scope of this manuscript. To address this point, we will clarify that the results do not show that localized transcripts are required, only that the transcripts are required somewhere specifically in the glial cell (without affecting the neuron level), and we can indeed show in an independent experiment that there are localized transcripts.

    Reviewer #2 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

    Major points:

    __ The authors analyse the 1700 shortlisted genes for Gene Ontology and associations with austism spectrum disorder, leading to interesting results. However, it is not clear to what extent the enrichments they observe are driven by their presumptive localization or if the associations are driven to a significant extent by the presence of these genes in the selected cell types in the Fly Cell Atlas. One way to address this would be to perform the GO and SFARI analysis on genes that are expressed in the same cells in the Fly Cell Atlas but were not shortlisted from the mammalian cell datasets - the results could then be compared to those obtained with the 1700 localized transcripts. __* This is a fair point raised by the reviewer as genes involved in neurological disease such as Autism Spectrum Disorder may be enriched in CNS/PNS cell types. We will follow the reviewer’s suggestion to perform GO and SFARI gene enrichment analysis in genes that were not shortlisted for presumptive glial localisation.

    * Although the authors attempt to justify its inclusion, I'm not convinced why it was important to use the whole cell transcriptome of perisynaptic Schwann cells as part of the selection process for localizing transcripts. Including this dataset may reduce the power of the pipeline by including mRNAs that are not localized to protrusions. How many of the shortlisted 1700 genes, and how many of the 11 glial localized mRNAs in Table 5, would be lost if the whole cell transcriptome were excluded. More generally, what is the distribution of the 11 validated localizing transcripts in each dataset in Table 4? This information might be valuable for determining which dataset(s), if any, has the best predictive power in this context. *

    We thank the reviewer for raising this point, which we will address with further analysis and adding to the discussion. We propose to address the criticism by running our analysis pipeline without the inclusion of the dataset using Perisynaptic Schwann Cells (PSCs) and then intersect with the PSCs-expressed genes, since their functional similarity with polarised Drosophila glial cells is highly relevant. We also agree with the reviewer that it would be a useful control for us to assess the ‘predictive power’ of each glial dataset by calculating their contribution to the shortlisted 1,700 glial localised transcripts and to the 11 experimentally validated transcripts via in situ hybridisation. To address this point, we plan to add this information in the revised manuscript.

    __ Did the authors check if any of the RNAi constructs are reducing levels of the target mRNA or protein? Doing so would strengthen the confidence in these important results significantly. In any case, the authors should also mention the caveat of potential off-target effects of RNAi. __*

    We thank the reviewer for their useful comment and agree that the extent to which the RNAi expression reduces the levels of mRNA is not specifically known. We will add a FISH experiment on lac, pdi and gs2 RNAi showing very strong reduction in mRNA levels. We will also add an explanation of the caveats of the use of the RNAi system to the discussion.

    * Methods: what is the justification for assuming that if the RNAi cross caused embryonic or larval lethality then the 'next most suitable' RNAi line is reporting on a phenotype specific to the gene. If the authors want to claim the effect is associated with different degrees of knockdown they should show this experimentally. An alternative explanation is that the line used for phenotypic analysis in glia is associated with an off-target effect. *

    We thank the reviewer for this comment. We agree that off target effects cannot in principle be completely ruled out without considerable additional experimental analysis beyond the scope of this manuscript. To address the criticism we will remove the expression data of the lines that cause lethality and revise the discussion to explain that the level of knockdown in each line is unknown, and would require further experimental exploration.

    Minor points:

    1. * It would be helpful to have in the Introduction (rather than the Results, as is currently the case) an operational definition of mRNA localization in the context of the study. And is it known whether or not localization in protrusions is the norm in mammalian glia or the Drosophila larval glia? I ask because it may be that almost all mRNAs diffuse into the protrusion, so this is not a selective process. One interesting approach to test this idea might be to test if the 1700 shortlisted transcripts have a significant underrepresentation of 'housekeeping' functions. * We thank the reviewer for this excellent suggestion. To address the comment, we will move our explanation of the operational definition of mRNA localization to the Introduction. We will also perform enrichment analysis of housekeeping genes within 1,700 shortlisted transcripts compared to the transcriptome background, as the reviewer suggested.

    Reviewer #3 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

    Major points:

    1. * The authors have pooled data from different studies across different type of glial cells performed from in vitro to in vivo. While pooling datasets may reveal common transcripts enriched in processes, this may not be the best approach considering these are completely different types of glial cells with distinct function in neuronal physiology. * We thank the reviewer for highlighting the need for us to further justify why we pooled datasets. We will revise the manuscript to better emphasise that the overarching goal of our study was to try to discern a common set of localised transcripts shared between the cells. The problem with analysing and comparing individual data sets is that much of the variation may be due to differences in the methods used and amount of material, rather than differences in the type of cells used. We will revise the discussion to make this point and plan to explain that our approach corresponds well with a previous publication pooling localised mRNA datasets in neurons (von Kugelgen & Chekulaeva 2021).

    von Kuegelgen & Chekulaeva, 2020 DOI: 10.1002/wrna.1590

    * It is important to note the limitations of the study. For example, DeSeq2 is biased for highly expressed transcripts. How robust was the prediction for low abundance transcripts? *

    The presented 1,700 transcripts were shortlisted based on their presence and expression level (TPM) in glial protrusions rather than their relative enrichment. Nevertheless, the reviewer makes a valid criticism of our use of DESeq2, where we compared enriched transcripts in glial and neuronal protrusions in Figure 1D. To address this point we will discuss this caveat in the relevant section.

    The issue raised regarding low abundance transcript prediction raises an important question: does the likelihood of localisation to cell extremities correlate with mRNA abundance? We have already partially addressed this point, since our analysis of the fraction of localised transcripts per expression level quantiles shows only limited correlation. To address this comment, we will add these results in the revised manuscript as a supplementary figure.

    * The authors identify 1,700 transcripts that they classify as "predicted to be present" in the projections of the Drosophila PNS glia. This was based on the comparison to all the mammalian glial transcripts. Since the authors have access to a transcriptomic study from Perisynaptic Schwann cells (PSCs), the nonmyelinating glia associated with the NMJ isolated from mice; it would be more convincing to then validate the extent of overlap between Drosophila peripheral glial with the mammalian PSCs. This may reveal conserved features of localized transcripts in the PNS, particularly associated with the NMJ function. *

    Thank you for the valuable suggestion. A similar point was also raised by __[Reviewer #2 - Major point 2] __to re-run our pipeline excluding the PSCs dataset and intersect with the PSC transcriptome post-hoc. Please see the above section for our detailed response.

    * Fig 2: What is the extent of overlap between the translating fractions versus the localized fraction? It will be informative to perform the functional annotation of the translating glial transcripts as identified from Fig 1D. *

    This is an interesting question. To address this point, we plan to: (i) compare transcripts that are translated vs. localised in glial protrusions, and (ii) perform functional annotation enrichment analysis on the translated fraction of genes.

    * "We conclude predicted group of 1,700 are highly likely to be peripherally localized in Drosophila cytoplasmic glial projections". To validate their predictions, the authors test some of these candidates in only one glial cell type. It might be worthy to extend this for other differentially expressed genes localized in another glial type as well. *

    The presented in vivo analyses made use of the repo-GAL4 driver, which is active in all glial subtypes, including subperineurial, perineurial and wrapping glia that make distal projection to the larval neuromuscular junction. We agree that subtype-specific analysis would be highly informative, but we believe this is outside the scope of the current work where we aimed to identify conserved localised transcriptomes across all glial subtypes. Nevertheless, to address the comment, we plan to further clarify our use of pan-glial repo-GAL4 driver in the Results and Method section of the revised manuscript.

    * Figure 5: The authors perform KD of candidate transcripts to test the effect on synapse formation. However, these are KD with RNAi that spans across the entire cell. To make the claim about the importance of "target" RNA localization in glia stronger, ideally, they should disrupt the enrichment specifically in the glial protusions and test the impact on bouton formation. Do these three RNAs have any putative localization elements? *

    We agree with the review, that we would ideally test the effect of disruption of mRNA localization (and therefore localised translation). However, we feel these experiments are beyond the scope of this current study, as they will require a long road of defining localisation signals that are small enough to disrupt without affecting other functions. To address this comment we will revise the Discussion section to mention those difficulties explicitly, and clarify the limitations of the approach used in our study for greater transparency.

    Reviewer #4 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

    Major points:

    1. * The authors use FISH to validate the glial expression of their target genes, though these experiments are not quantified, and no controls are shown. The authors should provide a supplemental figure with "no probe" controls, and/or validate the specificity of the probe via glial knockdown of the target gene (see point 2). Furthermore, these data should be quantified (e.g. number of puncta colocalized with NMJ glia membrans). * Thank you for requesting further information regarding the YFP smFISH probes. We have validated the specificity and sensitivity of the YFP probe in our recent publication (Titlow et al, 2023, Figure 1 and S1). Specifically, we demonstrated the lack of YFP probe signal from wild-type untagged biosamples and showed colocalization of YFP spots with additional probes targeting the endogenous exon of the transcript. Nevertheless, we will address this comment by adding control image panels of smFISH in wild-type (OrR) neuromuscular junction preparations.

    Titlow et al, 2023 DOI: 10.1083/jcb.202205129

    * *

    * For the most part, the authors only use one RNAi line for their functional studies, and they only show data for one line, even if multiple were used. To rule out potential false negatives, the authors should leverage their FISH probes to show the efficacy of their knockdowns in glia. This would serve the dual purpose of validating the new probes (see point 1). *

    Thank you for the suggestion. This point was also raised by [Reviewer #2 - Major point 3]. Please see above for our detailed response.

    * In Figure 5 E, given the severe reduction in size in the stimulated Pdi KD animals, the authors should show images of the unstimulated nerve as well. Do the nerve terminals actually shrink in size in these animals following stimulation, rather than expand? The NMJ looks substantially smaller than a normal L3 NMJ, though their quantification of neurite size in F suggests they're normal until stimulation. *

    We share the same interpretation of the data with the reviewer that the neurite area is reduced post-potassium stimulation in pdi knockdown animals. We will follow the reviewer’s suggestion and add an image showing unstimulated neuromuscular junctions.

    Minor points:

    * The authors claim that there is an enrichment of ASD-related genes in their final list of ~1400 genes that are enriched in glial processes. It is well-appreciated that synaptically-localized mRNAs are generally linked to ASDs. Can the authors comment on whether the transcripts localized to glial processes are even more linked to ASDs and neurological disorders than transcripts known to be localized to neuronal processes? *

    * *

    This is an interesting point. To address the comment, we will add a comparison of the degree of enrichment of ASD-related genes in neurite vs. glial protrusions in the revised manuscript.

    __*

    *__

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

    Reviewer #1

    1. * The use of blue/green or blue/green/magenta is difficult to resolve in some places. Swapping blue for cyan would greatly aid in visualizing their data. *

    This comment is much appreciated. We have swapped blue for cyan in Figures 4 and S4. We have also changed Figure S1 to increase contrast and visibility as per reviewer’s comment.

    * Make the colouring/formatting of the tables more consistent, its distracting when its constantly changing (also there is no need for a blue background.. just use a basic white table). *

    This comment is much appreciated. We have applied a consistent colour palette to the Tables without background colourings and made the formatting uniform.

    Reviewer #2

    * Introduction: 'Asymmetric mRNA localization is likely to be as important in glia, as it is in neurons,...'. Remove commas *

    Thank you for pointing this mistake out. We have made the corresponding edits.

    Reviewer #3

    * RNA localization in oligodendrocytes has been well studied and characterized. The authors should cite and discuss those papers (PMID: 18442491; PMID: 9281585).*

    We thank the reviewer for this useful suggestion. We have added these references to the paper.

    Reviewer #4

    1. * In Figure 5D, the authors should include a label to indicate that these images are from an unstimulated condition. * We thank the reviewer for pointing this out. We have added the label as requested.

    * The authors are missing a number of key citations for studies that have explored the functional significance of mRNA trafficking in glia, and those that have validated activity-dependent translation: *

    - ____https://pubmed.ncbi.nlm.nih.gov/18490510____*/ *

    -____https://pubmed.ncbi.nlm.nih.gov/7691830____*/ *

    -____https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3001053

    -____https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7450274____/

    __-____https://pubmed.ncbi.nlm.nih.gov/____36261025____*/

    *__

    We thank the reviewer for the comment. We have added these references to the text.

    4. Description of analyses that authors prefer not to carry out

  6. Review Commons

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

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    Reply to the reviewers

    1. General Statements

    We thank all four reviewers for their helpful and constructive comments. We have gone through each and every comment and proposed how we would address each point raised by the reviewers. We are confident our proposed revisions are feasible within a reasonable and expected time frame. Some of the comments regarding minor typo/aesthetics and extra references have already been addressed in the transferred manuscript. The changes are highlighted in yellow in the transferred manuscript.

    2. Description of the planned revisions

    Reviewer #1

    Major points:

    1. The presented work itself (Figures 1-4) does not need significant adjustments prior to publication, in my view, with only a few points to address. However, the work in Figure 5- doesn't really support the claims the authors make on its own, and would require some additional experiments or at the very least discussion of the caveats to its current form.

    We thank the reviewer for these comments and will follow the reviewer’s suggestion by discussing the caveats regarding the interpretation of Figure 5. We will also add to the discussion to suggest future research approaches beyond the scope of this manuscript that would address the functional importance of localised mRNA translation. We will briefly mention in the discussion methods such as the quantification of the mRNA foci and the disruption of the mRNA localisation signals to disrupt localised translation and the use of techniques such as Sun-Tag (Tanenbaum et al, 2014) and FLARIM (Richer et al, 2021) to visualise local translation directly.

    Tanenbaum et al, 2014 DOI: 10.1016/j.cell.2014.09.039

    Richer et al, 2021 DOI: 10.1101/2021.08.13.456301

    1. Localized glia transcripts, are they "glial/CNS/PNS" significant or are they similar to other known datasets of protrusion transcriptomes? The authors compared their 4801 "total" localized to a local transcriptome dataset from the Chekulaeva lab finding that a significant fraction are localized in both. As the authors note, this is in good agreement with a recent paper from the Talifarro lab showing conservation of localization of mRNAs across different cell types. What the authors haven't done here, is further test this by looking at other non-neuronal projection transcriptomic datasets (for example Mardakheh Developmental Cell 2015, among others). If the predicted glia-localized processes are similar to non-neuronal processes transcriptomes, this would further strengthen this claim and rule out some level of CNS/PNS derived linage driving the similarities between glia and neuronal localized transcripts.

    This is a good point and we thank the review for pointing out this interesting cancer data set. We will do as the reviewer suggests and intersect our data with Mardakheh Dev Cell 2015 to test the further generality of localisation in neurons and glia, in other cell types. Specifically, we plan to intersect both glial (this study) and neuronal (von Kuegelgen & Chekulaeva, 2020) dataset with protrusive breast cancer cells (Mardakeh et al, 2015).

    von Kuegelgen & Chekulaeva, 2020 DOI: 10.1002/wrna.1590

    Mardakeh et al, 2015 DOI: 10.1016/j.devcel.2015.10.005

    1. The presentation/discussion around Figure 3 is a bit weaker than other parts of the manuscript, and it doesn't really contribute to the story in its current form. Notably there is no discussion about the significance of glia in neurological disorders until the very end of the manuscript (page 21), meaning when its first brought up.. it just sits there as a one off side point. The authors might consider strengthening/tightening up the discussion here, if they really want to keep it as a solo main figure rather than integrating it somewhere else/putting it into supplemental. In my view, Figures 2 & 3 should be merged into something a bit more streamlined.

    This is a good point. We plan to strengthen the presentation of Figure 3 and discussion of the significance of glia in neurological disorders by adding a description of the Figure in the Results section and highlighting the significance of glia in nervous system disorders in the Discussion section.

    1. Why aren't there more examples of different mRNAs in Figure 4? Seems a waste to kick them all to supplemental.

    We agree that it could be helpful to show different expression patterns in the main figure. To address this point we will add Pdi (Fig. S4D), which shows mRNA expression in both the glia and the surrounding muscle cell. This pattern is in contrast to Gs2, which is highly specific to glial cells. We will also note that although pdi mRNA is present in both the glia and muscle, Pdi protein is only abundant in the glia, suggesting that translation of pdi mRNA to protein is regulated in a cell-specific manner.

    1. The plasticity experiments, while creative, I think need to be approached far more cautiously in their interpretation. Given that the siRNAs will completely deplete these mRNAs- it really needs to be stressed any/all of the effects seen could just be the result of "defective" or "altered" states in this glial population- which has spill over effects on plasticity in at the NMJ. Without directly visualizing if these mRNAs are locally translated in these processes and assessing if their translation is modulated by their plasticity paradigm, all these experiments can say is that these RNAs are needed in glia to modulate ghost bouton formation in axons. This represents the weakest part of this manuscript, and the part that I feel does not actually backup the claims currently being made. Without any experiments to A. quantify how much of these transcripts are localized vs in the cell body of these glia, B. visualize/quantify the translation of these mRNAs during baseline and during plasticity; the authors cannot use these data to claim that localized mRNAs are required for synaptic plasticity.

    We are grateful to the reviewer for pointing out that we were not precise enough in defining our interpretation of the structural plasticity assay. We did not intend to claim that our results show that local translation of these transcripts is necessary for plasticity, only that these transcripts are localized and are required in the glia for plasticity in the adjacent neuron (in which the transcript levels are not disrupted in the experiment). Definitively proving that these transcripts are required locally and translated in response to synaptic activity would require genetic/chemical perturbations and imaging assays that would require a year or more to complete, so are beyond the scope of this manuscript. To address this point, we will clarify that the results do not show that localized transcripts are required, only that the transcripts are required somewhere specifically in the glial cell (without affecting the neuron level), and we can indeed show in an independent experiment that there are localized transcripts.

    Reviewer #2

    Major points:

    1. The authors analyse the 1700 shortlisted genes for Gene Ontology and associations with austism spectrum disorder, leading to interesting results. However, it is not clear to what extent the enrichments they observe are driven by their presumptive localization or if the associations are driven to a significant extent by the presence of these genes in the selected cell types in the Fly Cell Atlas. One way to address this would be to perform the GO and SFARI analysis on genes that are expressed in the same cells in the Fly Cell Atlas but were not shortlisted from the mammalian cell datasets - the results could then be compared to those obtained with the 1700 localized transcripts.

    This is a fair point raised by the reviewer as genes involved in neurological disease such as Autism Spectrum Disorder may be enriched in CNS/PNS cell types. We will follow the reviewer’s suggestion to perform GO and SFARI gene enrichment analysis in genes that were not shortlisted for presumptive glial localisation.

    1. Although the authors attempt to justify its inclusion, I'm not convinced why it was important to use the whole cell transcriptome of perisynaptic Schwann cells as part of the selection process for localizing transcripts. Including this dataset may reduce the power of the pipeline by including mRNAs that are not localized to protrusions. How many of the shortlisted 1700 genes, and how many of the 11 glial localized mRNAs in Table 5, would be lost if the whole cell transcriptome were excluded. More generally, what is the distribution of the 11 validated localizing transcripts in each dataset in Table 4? This information might be valuable for determining which dataset(s), if any, has the best predictive power in this context.

    We thank the reviewer for raising this point, which we will address with further analysis and adding to the discussion. We propose to address the criticism by running our analysis pipeline without the inclusion of the dataset using Perisynaptic Schwann Cells (PSCs) and then intersect with the PSCs-expressed genes, since their functional similarity with polarised Drosophila glial cells is highly relevant. We also agree with the reviewer that it would be a useful control for us to assess the ‘predictive power’ of each glial dataset by calculating their contribution to the shortlisted 1,700 glial localised transcripts and to the 11 experimentally validated transcripts via in situ hybridisation. To address this point, we plan to add this information in the revised manuscript.

    1. Did the authors check if any of the RNAi constructs are reducing levels of the target mRNA or protein? Doing so would strengthen the confidence in these important results significantly. In any case, the authors should also mention the caveat of potential off-target effects of RNAi.

    We thank the reviewer for their useful comment and agree that the extent to which the RNAi expression reduces the levels of mRNA is not specifically known. We will add a FISH experiment on lac, pdi and gs2 RNAi showing very strong reduction in mRNA levels. We will also add an explanation of the caveats of the use of the RNAi system to the discussion.

    1. Methods: what is the justification for assuming that if the RNAi cross caused embryonic or larval lethality then the 'next most suitable' RNAi line is reporting on a phenotype specific to the gene. If the authors want to claim the effect is associated with different degrees of knockdown they should show this experimentally. An alternative explanation is that the line used for phenotypic analysis in glia is associated with an off-target effect.

    We thank the reviewer for this comment. We agree that off target effects cannot in principle be completely ruled out without considerable additional experimental analysis beyond the scope of this manuscript. To address the criticism we will remove the expression data of the lines that cause lethality and revise the discussion to explain that the level of knockdown in each line is unknown, and would require further experimental exploration.

    Minor points:

    1. It would be helpful to have in the Introduction (rather than the Results, as is currently the case) an operational definition of mRNA localization in the context of the study. And is it known whether or not localization in protrusions is the norm in mammalian glia or the Drosophila larval glia? I ask because it may be that almost all mRNAs diffuse into the protrusion, so this is not a selective process. One interesting approach to test this idea might be to test if the 1700 shortlisted transcripts have a significant underrepresentation of 'housekeeping' functions.

    We thank the reviewer for this excellent suggestion. To address the comment, we will move our explanation of the operational definition of mRNA localization to the Introduction. We will also perform enrichment analysis of housekeeping genes within 1,700 shortlisted transcripts compared to the transcriptome background, as the reviewer suggested.

    Reviewer #3

    Major points:

    1. The authors have pooled data from different studies across different type of glial cells performed from in vitro to in vivo. While pooling datasets may reveal common transcripts enriched in processes, this may not be the best approach considering these are completely different types of glial cells with distinct function in neuronal physiology.

    We thank the reviewer for highlighting the need for us to further justify why we pooled datasets. We will revise the manuscript to better emphasise that the overarching goal of our study was to try to discern a common set of localised transcripts shared between the cells. The problem with analysing and comparing individual data sets is that much of the variation may be due to differences in the methods used and amount of material, rather than differences in the type of cells used. We will revise the discussion to make this point and plan to explain that our approach corresponds well with a previous publication pooling localised mRNA datasets in neurons (von Kugelgen & Chekulaeva 2021).

    von Kuegelgen & Chekulaeva, 2020 DOI: 10.1002/wrna.1590

    1. It is important to note the limitations of the study. For example, DeSeq2 is biased for highly expressed transcripts. How robust was the prediction for low abundance transcripts?

    The presented 1,700 transcripts were shortlisted based on their presence and expression level (TPM) in glial protrusions rather than their relative enrichment. Nevertheless, the reviewer makes a valid criticism of our use of DESeq2, where we compared enriched transcripts in glial and neuronal protrusions in Figure 1D. To address this point we will discuss this caveat in the relevant section.

    The issue raised regarding low abundance transcript prediction raises an important question: does the likelihood of localisation to cell extremities correlate with mRNA abundance? We have already partially addressed this point, since our analysis of the fraction of localised transcripts per expression level quantiles shows only limited correlation. To address this comment, we will add these results in the revised manuscript as a supplementary figure.

    1. The authors identify 1,700 transcripts that they classify as "predicted to be present" in the projections of the Drosophila PNS glia. This was based on the comparison to all the mammalian glial transcripts. Since the authors have access to a transcriptomic study from Perisynaptic Schwann cells (PSCs), the nonmyelinating glia associated with the NMJ isolated from mice; it would be more convincing to then validate the extent of overlap between Drosophila peripheral glial with the mammalian PSCs. This may reveal conserved features of localized transcripts in the PNS, particularly associated with the NMJ function.

    Thank you for the valuable suggestion. A similar point was also raised by [Reviewer #2 - Major point 2] to re-run our pipeline excluding the PSCs dataset and intersect with the PSC transcriptome post-hoc. Please see the above section for our detailed response.

    1. Fig 2: What is the extent of overlap between the translating fractions versus the localized fraction? It will be informative to perform the functional annotation of the translating glial transcripts as identified from Fig 1D.

    This is an interesting question. To address this point, we plan to: (i) compare transcripts that are translated vs. localised in glial protrusions, and (ii) perform functional annotation enrichment analysis on the translated fraction of genes.

    1. "We conclude predicted group of 1,700 are highly likely to be peripherally localized in Drosophila cytoplasmic glial projections". To validate their predictions, the authors test some of these candidates in only one glial cell type. It might be worthy to extend this for other differentially expressed genes localized in another glial type as well.

    The presented in vivo analyses made use of the repo-GAL4 driver, which is active in all glial subtypes, including subperineurial, perineurial and wrapping glia that make distal projection to the larval neuromuscular junction. We agree that subtype-specific analysis would be highly informative, but we believe this is outside the scope of the current work where we aimed to identify conserved localised transcriptomes across all glial subtypes. Nevertheless, to address the comment, we plan to further clarify our use of pan-glial repo-GAL4 driver in the Results and Method section of the revised manuscript.

    1. Figure 5: The authors perform KD of candidate transcripts to test the effect on synapse formation. However, these are KD with RNAi that spans across the entire cell. To make the claim about the importance of "target" RNA localization in glia stronger, ideally, they should disrupt the enrichment specifically in the glial protusions and test the impact on bouton formation. Do these three RNAs have any putative localization elements?

    We agree with the review, that we would ideally test the effect of disruption of mRNA localization (and therefore localised translation). However, we feel these experiments are beyond the scope of this current study, as they will require a long road of defining localisation signals that are small enough to disrupt without affecting other functions. To address this comment we will revise the Discussion section to mention those difficulties explicitly, and clarify the limitations of the approach used in our study for greater transparency.

    Reviewer #4

    Major points:

    1. The authors use FISH to validate the glial expression of their target genes, though these experiments are not quantified, and no controls are shown. The authors should provide a supplemental figure with "no probe" controls, and/or validate the specificity of the probe via glial knockdown of the target gene (see point 2). Furthermore, these data should be quantified (e.g. number of puncta colocalized with NMJ glia membrans).

    Thank you for requesting further information regarding the YFP smFISH probes. We have validated the specificity and sensitivity of the YFP probe in our recent publication (Titlow et al, 2023, Figure 1 and S1). Specifically, we demonstrated the lack of YFP probe signal from wild-type untagged biosamples and showed colocalization of YFP spots with additional probes targeting the endogenous exon of the transcript. Nevertheless, we will address this comment by adding control image panels of smFISH in wild-type (OrR) neuromuscular junction preparations.

    Titlow et al, 2023 DOI: 10.1083/jcb.202205129

    1. For the most part, the authors only use one RNAi line for their functional studies, and they only show data for one line, even if multiple were used. To rule out potential false negatives, the authors should leverage their FISH probes to show the efficacy of their knockdowns in glia. This would serve the dual purpose of validating the new probes (see point 1).

    Thank you for the suggestion. This point was also raised by [Reviewer #2 - Major point 3]. Please see above for our detailed response.

    1. In Figure 5 E, given the severe reduction in size in the stimulated Pdi KD animals, the authors should show images of the unstimulated nerve as well. Do the nerve terminals actually shrink in size in these animals following stimulation, rather than expand? The NMJ looks substantially smaller than a normal L3 NMJ, though their quantification of neurite size in F suggests they're normal until stimulation.

    We share the same interpretation of the data with the reviewer that the neurite area is reduced post-potassium stimulation in pdi knockdown animals. We will follow the reviewer’s suggestion and add an image showing unstimulated neuromuscular junctions.

    Minor points:

    1. The authors claim that there is an enrichment of ASD-related genes in their final list of ~1400 genes that are enriched in glial processes. It is well-appreciated that synaptically-localized mRNAs are generally linked to ASDs. Can the authors comment on whether the transcripts localized to glial processes are even more linked to ASDs and neurological disorders than transcripts known to be localized to neuronal processes?

    This is an interesting point. To address the comment, we will add a comparison of the degree of enrichment of ASD-related genes in neurite vs. glial protrusions in the revised manuscript.

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

    Reviewer #1

    1. The use of blue/green or blue/green/magenta is difficult to resolve in some places. Swapping blue for cyan would greatly aid in visualizing their data.

    This comment is much appreciated. We have swapped blue for cyan in Figures 4 and S4. We have also changed Figure S1 to increase contrast and visibility as per reviewer’s comment.

    1. Make the colouring/formatting of the tables more consistent, its distracting when its constantly changing (also there is no need for a blue background.. just use a basic white table).

    This comment is much appreciated. We have applied a consistent colour palette to the Tables without background colourings and made the formatting uniform.

    Reviewer #2

    1. Introduction: 'Asymmetric mRNA localization is likely to be as important in glia, as it is in neurons,...'. Remove commas

    Thank you for pointing this mistake out. We have made the corresponding edits.

    Reviewer #3

    1. RNA localization in oligodendrocytes has been well studied and characterized. The authors should cite and discuss those papers (PMID: 18442491; PMID: 9281585).

    We thank the reviewer for this useful suggestion. We have added these references to the paper.

    Reviewer #4

    1. In Figure 5D, the authors should include a label to indicate that these images are from an unstimulated condition.

    We thank the reviewer for pointing this out. We have added the label as requested.

    1. The authors are missing a number of key citations for studies that have explored the functional significance of mRNA trafficking in glia, and those that have validated activity-dependent translation:

    - https://pubmed.ncbi.nlm.nih.gov/18490510/

    -https://pubmed.ncbi.nlm.nih.gov/7691830/

    -https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3001053

    -https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7450274/

    -https://pubmed.ncbi.nlm.nih.gov/36261025**_/

    _**

    We thank the reviewer for the comment. We have added these references to the text.

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    Referee #4

    Evidence, reproducibility and clarity

    Peripheral localization of mRNAs to cellular protrusions (e.g. axons, astrocyte peri-synaptic processes, myelin sheaths) is important for activity-dependent regulation of nervous system function. In Gala et al., the authors aim to identify conserved, peripherally located mRNAs in through a combination of unbiased transcriptomics and in vivo validation in Drosophila NMJ glia. To that end, they mine several published datasets enriched for peripherally located mRNAs in glia, identify which of these transcripts are conserved in fly, filtered for mRNAs that are enriched in processes over soma, and filtered for mRNAs that were enriched in three glial subtypes that ensheath their model system: Drosophila NMJ. Finally, they go on to validate 11 (of 15) predicted genes as located in NMJ glia, demonstrating that loss of these transcripts, in some cases, impacts synaptic plasticity.
    This is an interesting study that complements a growing interest in the community: how do glia locally support the neurons that interact with. I have a number of suggestions to support the conclusions made in this manuscript:

    Major:

    1. The authors use FISH to validate the glial expression of their target genes, though these experiments are not quantified, and no controls are shown. The authors should provide a supplemental figure with "no probe" controls, and/or validate the specificity of the probe via glial knockdown of the target gene (see point 2). Furthermore, these data should be quantified (e.g. number of puncta colocalized with NMJ glia membrans).
    2. For the most part, the authors only use one RNAi line for their functional studies, and they only show data for one line, even if multiple were used. To rule out potential false negatives, the authors should leverage their FISH probes to show the efficacy of their knockdowns in glia. This would serve the dual purpose of validating the new probes (see point 1).
    3. In Figure 5 E, given the severe reduction in size in the stimulated Pdi KD animals, the authors should show images of the unstimulated nerve as well. Do the nerve terminals actually shrink in size in these animals following stimulation, rather than expand? The NMJ looks substantially smaller than a normal L3 NMJ, though their quantification of neurite size in F suggests they're normal until stimulation.

    Minor:

    1. In Figure 5 D, the authors should include a label to indicate that these images are from an unstimulated condition.
    2. The authors claim that there is an enrichment of ASD-related genes in their final list of ~1400 genes that are enriched in glial processes. It is well-appreciated that synaptically-localized mRNAs are generally linked to ASDs. Can the authors comment on whether the transcripts localized to glial processes are even more linked to ASDs and neurological disorders than transcripts known to be localized to neuronal processes?
    3. The authors are missing a number of key citations for studies that have explored the functional significance of mRNA trafficking in glia, and those that have validated activity-dependent translation:

    Significance

    Glia are morphologically complex cells that extend tens (microglia/oligodendrocytes) to thousands
    (astrocytes) of processes to interact with and support neurons. Given this complex morphology, and the ability of glia to dynamically respond to changes in neuronal activity, there has been a push in recent years to characterize local mechanisms of glia-neuron support. These mechanisms include mRNA trafficking and activity-dependent translation in distal processes. The authors of this study did a nice job computationally identifying a set of putative genes that are enriched in glial processes, which should be of broad interest to the glial community.

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    Referee #3

    Evidence, reproducibility and clarity

    In the manuscript by Gala et al, the authors perform meta-analysis of transcriptomic datasets from different studies focused on synaptically-associated mammalian glial cells. Based on a detailed analysis, the authors predict 1700 localized transcripts and attempted to identify the local transcriptome in glial cells conserved in Drosophila and mammals. This kind of data mining is informative, and can predict the top candidates relatively well, however, the study suffers from lack of depth and a careful assessment comparing across the different datasets. The main concerns have been listed below and suggestions for a more detailed an careful assessment of the data.

    1. The authors have pooled data from different studies across different type of glial cells performed from in vitro to in vivo. While pooling datasets may reveal common transcripts enriched in processes, this may not be the best approach considering these are completely different types of glial cells with distinct function in neuronal physiology.
    2. It is important to note the limitations of the study. For example, DeSeq2 is biased for highly expressed transcripts. How robust was the prediction for low abundance transcripts?
    3. The authors identify 1,700 transcripts that they classify as "predicted to be present" in the projections of the Drosophila PNS glia. This was based on the comparison to all the mammalian glial transcripts. Since the authors have access to a transcriptomic study from Perisynaptic Schwann cells (PSCs), the nonmyelinating glia associated with the NMJ isolated from mice; it would be more convincing to then validate the extent of overlap between Drosophila peripheral glial with the mammalian PSCs. This may reveal conserved features of localized transcripts in the PNS, particularly associated with the NMJ function.
    4. Fig 2: What is the extent of overlap between the translating fractions versus the localized fraction? It will be informative to perform the functional annotation of the translating glial transcripts as identified from Fig 1D.
    5. "We conclude predicted group of 1,700 are highly likely to be peripherally localized in Drosophila cytoplasmic glial projections". To validate their predictions, the authors test some of these candidates in only one glial cell type. It might be worthy to extend this for other differentially expressed genes localized in another glial type as well.
    6. Figure 5: The authors perform KD of candidate transcripts to test the effect on synapse formation. However, these are KD with RNAi that spans across the entire cell. To make the claim about the importance of "target" RNA localization in glia stronger, ideally, they should disrupt the enrichment specifically in the glial protusions and test the impact on bouton formation. Do these three RNAs have any putative localization elements?
    7. RNA localization in oligodendrocytes has been well studied and characterized. The authors should cite and discuss those papers (PMID: 18442491; PMID: 9281585).

    Significance

    In the manuscript by Gala et al, the authors perform meta-analysis of transcriptomic datasets from different studies focused on synaptically-associated mammalian glial cells. Based on a detailed analysis, the authors predict 1700 localized transcripts and attempted to identify the local transcriptome in glial cells conserved in Drosophila and mammals. This kind of data mining is informative, and can predict the top candidates relatively well, however, the study suffers from lack of depth and a careful assessment comparing across the different datasets and the biological significance.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    The manuscript by Gala et al. describes efforts to systematically identify candidate localizing mRNAs within glial cells of the Drosophila larval nervous system. They address this issue with an interesting and novel approach that involves compiling candidate localized transcripts from available mammalian cell datasets and cross-referencing a list of mRNAs from homologous Drosophila genes with a small number of candidates for mRNA localization in Drosophila glia cells that this team identified in a previous study (which they have now validated further). The results provide compelling evidence that their pipeline has predictive power. The authors then go on to perform functional analysis of a small number of validated localizing transcripts and provide evidence that several of these genes are functionally important in glia, including in processes related to synaptic plasticity. These observations raise the possibility that the localization process is functionally important but this was not tested directly (doing so would require a separate long-term study).

    The manuscript is well written and the figures are of high quality. Methodological details are provided, as is quantification where appropriate. However, some points are not yet sufficiently strongly supported by the data yet and the justification for some aspects of the workflow needs clarifying.

    Major points:

    1. The authors analyse the 1700 shortlisted genes for Gene Ontology and associations with austism spectrum disorder, leading to interesting results. However, it is not clear to what extent the enrichments they observe are driven by their presumptive localization or if the associations are driven to a significant extent by the presence of these genes in the selected cell types in the Fly Cell Atlas. One way to address this would be to perform the GO and SFARI analysis on genes that are expressed in the same cells in the Fly Cell Atlas but were not shortlisted from the mammalian cell datasets - the results could then be compared to those obtained with the 1700 localized transcripts.
    2. Although the authors attempt to justify its inclusion, I'm not convinced why it was important to use the whole cell transcriptome of perisynaptic Schwann cells as part of the selection process for localizing transcripts. Including this dataset may reduce the power of the pipeline by including mRNAs that are not localized to protrusions. How many of the shortlisted 1700 genes, and how many of the 11 glial localized mRNAs in Table 5, would be lost if the whole cell transcriptome were excluded. More generally, what is the distribution of the 11 validated localizing transcripts in each dataset in Table 4? This information might be valuable for determining which dataset(s), if any, has the best predictive power in this context.
    3. Did the authors check if any of the RNAi constructs are reducing levels of the target mRNA or protein? Doing so would strengthen the confidence in these important results significantly. In any case, the authors should also mention the caveat of potential off-target effects of RNAi.
    4. Methods: what is the justification for assuming that if the RNAi cross caused embryonic or larval lethality then the 'next most suitable' RNAi line is reporting on a phenotype specific to the gene. If the authors want to claim the effect is associated with different degrees of knockdown they should show this experimentally. An alternative explanation is that the line used for phenotypic analysis in glia is associated with an off-target effect.

    Minor points:

    1. It would be helpful to have in the Introduction (rather than the Results, as is currently the case) an operational definition of mRNA localization in the context of the study. And is it known whether or not localization in protrusions is the norm in mammalian glia or the Drosophila larval glia? I ask because it may be that almost all mRNAs diffuse into the protrusion, so this is not a selective process. One interesting approach to test this idea might be to test if the 1700 shortlisted transcripts have a significant underrepresentation of 'housekeeping' functions.
    2. Introduction: 'Asymmetric mRNA localization is likely to be as important in glia, as it is in neurons,...'. Remove commas

    Significance

    Whilst glial mRNA localization has been reported in other systems, this study is significant as it paves the way for functional and mechanistic dissection of this process in a genetically tractable model system that involves physiologically relevant neuron-glia interactions. The successful results of the data mining approach may also encourage others to address other problems in this manner. The study would be more impactful if additional hits from the 1700 transcripts were validated for glial localization but the findings in the current manuscript are still important. The advance is more methodological than conceptual. The work will appeal to cell biology and systems biology audiences.

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    Referee #1

    Evidence, reproducibility and clarity

    The study of mRNA localization in glia has been largely overlooked in comparison to neurons. However, glia are also polarized cells with long cytoplasmic processes that play important roles in neural function. In this manuscript, Gala and Lee et al attempt to fill in this missing void in the literature. They first use a meta-analysis of existing, cross-species, transcriptomic data to identify a set of 1,700 transcripts that are likely to be localized to the periphery of glia in Drosophila. They then used this list of transcripts to predict which mammalian glial transcripts are also likely to be localized. They're analysis suggests that a large proportion of the mammalian glial transcripts are predicted to be localized to the periphery of glia, and that these transcripts are enriched for functions involved in membrane trafficking, cytoskeleton regulation, local translation, and cell-cell communication. A connection with cross-cellular communication (ie glia-glia, glia-neuron, glia-muscle) at the neuromuscular junction, prompting the authors to assess if some of these transcripts play a role in plasticity of at the NMJ. Using siRNA driver lines, that loss of some of their assessed transcripts prevent new synapse formation- suggestive of a role for mRNA localization and local protein synthesis in driving synaptic plasticity. These findings suggest that mRNA localization is a widespread phenomenon in glia, and that it plays an equally important role as the more widely studied neuronal mRNA localization.

    Key Points

    • 1,700 transcripts are predicted to be localized to the periphery of PNS glia in Drosophila at the NMJ.
    • A large proportion of the mammalian glial transcripts are predicted to be localized to the periphery of glia.
    • Localized transcripts are enriched for functions involved in membrane trafficking, cytoskeleton regulation, local translation, and cell-cell communication.
    • Localized glial transcripts may function in synaptic plasticity.

    Major comments

    Overall, the work presented within manuscript is well reasoned and supports the points the authors generally are trying to make. The work as is, does fall a little on the light side, resting on almost exclusive bioinformatic analysis with some limited validation. The presented work itself (Figures 1-4) does not need significant adjustments prior to publication, in my view, with only a few points to address. However, the work in Figure 5- doesn't really support the claims the authors make on its own, and would require some additional experiments or at the very least discussion of the caveats to its current form.

    1. Localized glia transcripts, are they "glial/CNS/PNS" significant or are they similar to other known datasets of protrusion transcriptomes? The authors compared their 4801 "total" localized to a local transcriptome dataset from the Chekulaeva lab finding that a significant fraction are localized in both. As the authors note, this is in good agreement with a recent paper from the Talifarro lab showing conservation of localization of mRNAs across different cell types. What the authors haven't done here, is further test this by looking at other non-neuronal projection transcriptomic datasets (for example Mardakheh Developmental Cell 2015, among others). If the predicted glia-localized processes are similar to non-neuronal processes transcriptomes, this would further strengthen this claim and rule out some level of CNS/PNS derived linage driving the similarities between glia and neuronal localized transcripts.
    2. The presentation/discussion around Figure 3 is a bit weaker than other parts of the manuscript, and it doesn't really contribute to the story in its current form. Notably there is no discussion about the significance of glia in neurological disorders until the very end of the manuscript (page 21), meaning when its first brought up.. it just sits there as a one off side point. The authors might consider strengthening/tightening up the discussion here, if they really want to keep it as a solo main figure rather than integrating it somewhere else/putting it into supplemental. In my view, Figures 2 & 3 should be merged into something a bit more streamlined.
    3. Why aren't there more examples of different mRNAs in Figure 4? Seems a waste to kick them all to supplemental.
    4. The plasticity experiments, while creative, I think need to be approached far more cautiously in their interpretation. Given that the siRNAs will completely deplete these mRNAs- it really needs to be stressed any/all of the effects seen could just be the result of "defective" or "altered" states in this glial population- which has spill over effects on plasticity in at the NMJ. Without directly visualizing if these mRNAs are locally translated in these processes and assessing if their translation is modulated by their plasticity paradigm, all these experiments can say is that these RNAs are needed in glia to modulate ghost bouton formation in axons. This represents the weakest part of this manuscript, and the part that I feel does not actually backup the claims currently being made. Without any experiments to A. quantify how much of these transcripts are localized vs in the cell body of these glia, B. visualize/quantify the translation of these mRNAs during baseline and during plasticity; the authors cannot use these data to claim that localized mRNAs are required for synaptic plasticity.

    Minor points:

    1. The use of blue/green or blue/green/magenta is difficult to resolve in some places. Swapping blue for cyan would greatly aid in visualizing their data.
    2. Make the colouring/formatting of the tables more consistent, its distracting when its constantly changing (also there is no need for a blue background.. just use a basic white table).

    Significance

    This work would be well received within the field, being at a time where increasing focus on mRNA localization and local protein synthesis are major players in glia along with the more widely studied neurons. Additionally, that mRNAs localized in glia are relevant to neurological disorders also comes at a time where increasingly, especially in neurodegenerative disorders, glia are believed to be drivers of the diseases as well- suggesting that dysregulation of local protein synthesis in glia may play a role. The major weakness of this work is that being largely bioinformatic (from existing datasets), a lot of this is speculative and no conclusive data is shown to demonstrate how/that this glia population is utilizing mRNA localization in protrusions to fuel local protein synthesis for a specific purpose (ie synaptic plasticity).

    This work would be of interested to those broadly interested in glial biology and mRNA localization.

    The reviewers background is in RNA localization and local protein synthesis in neurons.

  12. Arcadia Science

    15 out of 19 fly genes had high confidence mammalian homologues, so we report our comparison within these 15 genes (see Table 5).

    At what point in the ortholog assignment approach were these genes "lost"? If the DIOPT cutoff were set to 7 instead of 8, for example, would these 4 remaining "positive control" genes be recovered?

  13. Arcadia Science

    In conclusion, these results show that the localization of transcripts, extrapolated from vertebrates to Drosophila, were highly concordant, reliably predicting the presence of multiple mRNAs at the glial periphery and highlighting a possible evolutionary conservation of glial protrusion-localized transcriptome.

    The authors used differential expression between soma and periphery in mouse/rat data to try to identify genes localized to the periphery in Drosophila. Were the authors able to confirm enrichment of these predicted genes in the periphery? It's a bit difficult to tell based on the images, but it seems more that these genes are simply present at the periphery.

  14. Arcadia Science

    To confirm our prediction of mRNA localization, we utilized single molecule Fluorescence In Situ Hybridisation (smFISH) experiments and surveyed transcripts that were previously reported to be present in the Drosophila NMJ glia (Titlow et al., 2022). These smFISH experiments revealed that more than 70% of the localized transcripts matched our prediction, demonstrating utility of our dataset.

    I think it would be helpful to more clearly state the number of transcripts evaluated for localization using smFISH. The "70%" value could be misinterpreted to be with respect to the 1700 mRNAs predicted to be localized to Drosophila glia.

  15. Arcadia Science

    C) Identification of high confidence D. melanogaster orthologs of 4,801 genes that were detected in at least 8 datasets. DRSC Integrative Ortholog Prediction Tool (DIOPT) score of 8 was used as cut-off.

    I'm curious if more mappings of mouse to fly genes could be recovered if a newer approach were used. There are a lot of papers now using OrthoFinder (https://genomebiology.biomedcentral.com/articles/10.1186/s13059-019-1832-y) to identify orthologs between species, for example.

  16. Arcadia Science

    We found that 11 out of the 15 transcripts (73.33%) were predicted from our analysis of mammalian glial transcripts to localize to the glial periphery in Drosophila.

    It might be helpful to include this result in as a fourth column in Table 5.

  17. Arcadia Science

    This manuscript leverages publicly available data from vertebrate glial periphery transcriptomes, as well as Drosophila single-cell RNA-Seq data, to try to predict the localization of mRNAs in the periphery of glia in the fruit fly Drosophila melanogaster. The authors predict around 1700 transcripts they expect to be localized to glial projections in Drosophila and test their predictions using a subset of genes previously known to be expressed in Drosophila PNS glia. The authors also functionally perturb genes they identified as localized to glia and show that these genes play a role in glial function.

    This manuscript does a great job of leveraging publicly available datasets and databases of multiple types, such as Gene Ontology and Disease Ontology, as well as drawing insights from the biology of multiple organisms.

    I think the authors were able to demonstrate that some genes in their analysis were present in the periphery of Drosophila glia, and that those genes function in the nervous system. It's unclear to me whether these genes are truly differentially expressed in the periphery relative to the soma, as might be predicted based on the starting mouse and rat data. It will be exciting to see how well these predictions perform in other glial cell types in Drosophila or other protostomes, and how this kind of analysis might be extended to explore additional invertebrates.

  18. Version published to 10.1101/2022.11.30.518536v2 on bioRxiv
  19. Arcadia Science

    This manuscript leverages publicly available data from vertebrate glial periphery transcriptomes, as well as Drosophila single-cell RNA-Seq data, to try to predict the localization of mRNAs in the periphery of glia in the fruit fly Drosophila melanogaster. The authors predict around 1700 transcripts they expect to be localized to glial projections in Drosophila and test their predictions using a subset of genes previously known to be expressed in Drosophila PNS glia. The authors also functionally perturb genes they identified as localized to glia and show that these genes play a role in glial function.

    This manuscript does a great job of leveraging publicly available datasets and databases of multiple types, such as Gene Ontology and Disease Ontology, as well as drawing insights from the biology of multiple organisms.

    I think the authors were able to demonstrate that some genes in their analysis were present in the periphery of Drosophila glia, and that those genes function in the nervous system. It's unclear to me whether these genes are truly differentially expressed in the periphery relative to the soma, as might be predicted based on the starting mouse and rat data. It will be exciting to see how well these predictions perform in other glial cell types in Drosophila or other protostomes, and how this kind of analysis might be extended to explore additional invertebrates.

  20. Arcadia Science

    In conclusion, these results show that the localization of transcripts, extrapolated from vertebrates to Drosophila, were highly concordant, reliably predicting the presence of multiple mRNAs at the glial periphery and highlighting a possible evolutionary conservation of glial protrusion-localized transcriptome.

    The authors used differential expression between soma and periphery in mouse/rat data to try to identify genes localized to the periphery in Drosophila. Were the authors able to confirm enrichment of these predicted genes in the periphery? It's a bit difficult to tell based on the images, but it seems more that these genes are simply present at the periphery.

  21. Arcadia Science

    To confirm our prediction of mRNA localization, we utilized single molecule Fluorescence In Situ Hybridisation (smFISH) experiments and surveyed transcripts that were previously reported to be present in the Drosophila NMJ glia (Titlow et al., 2022). These smFISH experiments revealed that more than 70% of the localized transcripts matched our prediction, demonstrating utility of our dataset.

    I think it would be helpful to more clearly state the number of transcripts evaluated for localization using smFISH. The "70%" value could be misinterpreted to be with respect to the 1700 mRNAs predicted to be localized to Drosophila glia.

  22. Arcadia Science

    We found that 11 out of the 15 transcripts (73.33%) were predicted from our analysis of mammalian glial transcripts to localize to the glial periphery in Drosophila.

    It might be helpful to include this result in as a fourth column in Table 5.

  23. Arcadia Science

    15 out of 19 fly genes had high confidence mammalian homologues, so we report our comparison within these 15 genes (see Table 5).

    At what point in the ortholog assignment approach were these genes "lost"? If the DIOPT cutoff were set to 7 instead of 8, for example, would these 4 remaining "positive control" genes be recovered?

  24. Arcadia Science

    C) Identification of high confidence D. melanogaster orthologs of 4,801 genes that were detected in at least 8 datasets. DRSC Integrative Ortholog Prediction Tool (DIOPT) score of 8 was used as cut-off.

    I'm curious if more mappings of mouse to fly genes could be recovered if a newer approach were used. There are a lot of papers now using OrthoFinder (https://genomebiology.biomedcentral.com/articles/10.1186/s13059-019-1832-y) to identify orthologs between species, for example.

  26. Version published to 10.1101/2022.11.30.518536v1 on bioRxiv