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Reply to the reviewers
We thank the reviewers for their thoughtful comments. We were delighted the reviewers found our results “compelling”, “striking”, “well presented”, “implications exciting”, “excellent results! really nice!”, “this microscopy is beautiful!” and “translational-dependence (of mRNA localization) in a transcript-specific way without perturbing translation globally”, which is a “complete surprise, and opens exciting doors to investigate how translation leads to mRNA organization and its connection to **tissue development” and “may represent a new pathway of mRNA transport”.
We also appreciated the comments regarding the “wide appeal”, “broad readership of readers”, and “broad interest” the reviewers gave to our manuscript regarding its impact, and also the comments of “well-written (and) well-cited”.
We can address all the concerns raised by the reviewers. In addition to textual changes, we will add the following to the Results section:
- Additional quantitation of smFISH beyond Figure 2;
- Addition of a negative (uniformly distributed) mRNA control and its quantitation;
- Western blots for our ΔATG lines to determine what and how much protein is made.
- Unbiased nuclear masking.
Our specific responses are shown below, in blue.
Reviewer #1
**Major comments**
Fig. 1: Main and supplementary figures present smFISH signals for eight localized mRNAs, while in the results section authors describe that they analyzed twenty-five transcripts. Authors should explain the choice of transcripts presented in the paper.
We will include a panel in Fig. S1E to show every mRNA that we tested, and we will edit Table 1 to describe the observed subcellular localization.
We will edit the text, adding a few sentences to clarify, along the lines of: “O**ur survey revealed mRNAs with varying degrees of localization within epithelia that we divided into three classes: CeAJ/membrane localized, perinuclearly localized, and unlocalized (Fig. 1 and S1 and Table 1).” and “The rest of our tested mRNAs did not possess any evident subcellular localization at any of the analyzed embryonic stages/tissues and were not further investigated (Fig. S1E and Table 1).”
Moreover, smFISH signal of different localized mRNAs in epidermal cells was visualized at different stages (bean, comma or late comma), and authors did not comment what was the reason of such conditions. This may make transcripts localization results difficult to interpret, as further analysis showed that mRNA localization varied in a stage-specific manner.
We have clarified this point now in Figure legend 1: “Specific embryonic stages were selected for each transcript based on the highest degree of mRNA localization they exhibited.”
Did author used smFISH probes designed against endogenous mRNAs for all tested transcripts?
We did not. We clarify this point now in Materials and methods: “All probes were designed against the endogenous mRNA sequences except dlg-1 (some constructs), pkc-3, hmp-2, spc-1, let-805, and vab-10a, whose mRNA were detected with gfp probes in their corresponding transgenic lines (Table S2). An exception to this is Fig. S1A where we used probes against the endogenous dlg-1 mRNA.”.
Marking dlg-1 mRNA as dlg-1-gfp suggests that smFISH probe was specific for gfp transcript. Is it true? If yes, authors should compare localization of wild-type endogenous dlg-1 mRNA with that of the transcript encoding a fusion protein, to confirm that fusion does not affect mRNA localization.
Yes, in Fig. 1C we show smFISH for GFP (i.e., the tagged dlg-1 only). In Fig. S1A, we show smFISH against endogenous dlg-1. Tagged and endogenous dlg-1 mRNAs are both localized. We clarified this point in the main text: “Five of these transcripts were enriched at specific loci at or near the cell membrane: laterally and at the CeAJ for dlg-1 (Fig. 1C for endogenous/GFP CRISPR-tagged dlg-1::gfp mRNA and S1A for endogenous/non-tagged dlg-1 mRNA), (…)”. And in the Supplemental figure legend (Fig. S1A): “Endogenous/non-tagged dlg-1 mRNA shows CeAJ/membrane localization like its endogenous/GFP CRISPR-tagged counterpart.”
Fig. 2B: Authors conclude that at later stages of pharyngeal morphogenesis mRNA enrichment at the CeAJ decreased gradually in comparison to comma stage. Data do not show statistically significant decrease in ratio of localized mRNAs - for dlg-1: bean: 0.39{plus minus}0.09, comma: 0.29{plus minus}0.08, 1.5-fold: 0.30{plus minus}0.09; for ajm-1: bean: 0.36{plus minus}0.08, comma: 0.30{plus minus}0.05, 1.5-fold: 0.28{plus minus}0.09.
t-test (one-tailed) analysis revealed a significant difference between bean and comma stages for both dlg-1 and ajm-1 mRNAs. Statistical analysis and data will be provided.
Fig. 4: What was the difference between the first and the second __ΔATG transgenic line? Authors should analyze the size of the truncated DLG-1 protein that is expressed from the second Δ__ATG transgenic line that localizes to CeAJ. Knowing alternative ATGs and protein size may suggest domain composition of the truncated protein. This will allow to confront truncated protein localization with the results from.
We will perform a Western blot to determine the size and levels of proteins produced.
Fig. 5. Moreover, to prove that the localization of dlg-1 mRNA at the CeAJ is translation-dependent, additional experiment should be performed where transcripts localization will be analyzed in embryos treated with translation inhibitors such as cycloheximide (translation elongation inhibitor) and puromycin (that induces premature termination).
We believe this comment might refer to Fig. 4. If this is the case: drugs like cycloheximide and puromycin affect the translation of the whole transcriptome, whereas with our ΔATG experiment, we aimed to target the translation of one specific transcript and avoid secondary effects. Nevertheless, we understand Reviewer #1’s concern and will include a second experiment. In our hands, cycloheximide and puromycin have never worked in older embryos (it’s hard to get past the eggshell and into the embryo). Instead, we will use stress conditions, which induce a “ribosome drop-off” (Spriggs et al., 2010). Heat stress has been shown to decrease polysome occupancy (Arnold et al., 2014). We, therefore, have used heat-shock at 33°C for 30’, and the results are now shown in Fig. S4. These show the loss of RNA localization upon heat shock.
**Minor comments**
In the introduction section authors should emphasize the main goal and scientific significance of the paper.
We added this sentence to state the significance before summarizing the results: “To investigate the impact of mRNA localization during embryonic development, we conducted a single molecule fluorescence in situ hybridization (smFISH)-based survey (…)” and “Our data demonstrate that the dlg-1 UTRs are dispensable, whereas translation is required for localization, therefore providing an example of a translation-dependent mechanism for mRNA delivery in C. elegans.” To state the significance.
Fig 1A: It's hard to distinguish different colors on the schematics. Schematics presents intermediate filaments that are not included in the Table 1.
We modified Table 1 based on this and other reviewers’ comments.
Fig. 1C: dlg-1 transcript is marked as dlg-1-gfp on the left panel and dlg-1 on the right panel.
Corrected.
Fig. 2B: Axis labels and titles are not visible, larger font size should be used.
We will modify the graph (following Reviewer #2’s suggestion) and axes label and title sizes will be taken into account.
Fig. 5C: Enlarge the font size.
Will do.
Fig. S2: Embryonic stages should be marked on the figure for easier interpretation.
Added.
Reviewer #2
Major comments
Figure 2 requires a negative (or uniformly distributed) mRNA control for comparison. Figure 2C should be quantified. The plot quality should be improved, and appropriate statistical tests should be employed to strengthen the claimed findings.
We will add a negative control (jac-1 mRNA), and quantify Fig. 2C as well. Plots will be changed accordingly to the suggestion.
Most claims of perinuclear mRNA localization are difficult to see and not well supported visually or statistically. The usage of DAPI markers, membrane markers, 3D rendering, or a quantified metric would bolster this claim. Also, sax-7 is claimed to be perinuclear and elsewhere claimed to be uniform then used as a uniform control. Please explain or resolve these discrepancies more clearly.__
Regarding perinuclear mRNAs:
We are not trying to make a big statement out of these data as perinuclear (ER) localization of mRNAs coding for transmembrane/secreted proteins is well known. The aim of our study was to describe transcript localized at or in the proximity of the junction. However, we thought it was worth mentioning these examples of perinuclearly localized mRNAs (hmr-1, sax-7, and eat-20) for two reasons: scientific correctness – show accessory results that might be interesting for other scientists – and use as positive controls for our smFISH survey – these mRNAs were expected to localize perinuclearly for the reasons mentioned above. We will rewrite the text to make these points clearer.
Regarding sax-7 mRNA:
sax-7 mRNA localizes perinuclearly in sporadic instances (Fig S1C), but it is predominantly scattered throughout the cytoplasm (i.e., unlocalized). It presumably localizes perinuclearly in a translation-dependent manner as sax-7 codes for a transmembrane protein that would be targeted to the ER. We have described this ER-type of localization in the introduction and reiterated it partially in the first paragraph of the results. sax-7 UTRs are therefore presumably not responsible for subcellular localization, which would instead depend on a signal sequence. We will better clarify this point in the main text.
The major concern about the paper is the data display and interpretation of Figure 5C. I'm not comfortable with the approach the authors took of blurring out the nucleus. A more faithful practice would be to use an automated mask over DAPI staining or to quantify the entirety of the cell. If the entirety of the cell were quantified, one could still focus analysis on specific regions of relevance. The interpretations distinguishing membrane versus cytoplasmic localization (or mislocalization) are hard to differentiate in these images especially since they are lacking a membrane marker. The ability to make these distinctions forms the basis of Tocchini et al's two pathways of dlg-1 mRNA localization. These interpretations also heavily rely on how the image was processed through the different Z-stacks, and it's not clear to me how that was done. For example, the diffusion of mRNA in figure 5F and 5I are indistinguishable to my eye but are claimed to be different.
In the images, the nuclei have been blurred to allow the reader to focus on the cytoplasmic signal and not on the nuclear (transcriptional) signal as it is not meaningful for this study. In the quantitation, the nuclear signal has been unbiasedly and specifically removed from the analysis by cropping out the DNA signal from the other channels. The frontal plane views of the seam cells in Fig. 5 show maximum intensity projections (MIPs) of 3 Z-stacks (0.54 µm total) that each contain nuclei and, therefore, the transcriptional signal (schematics in Fig. 5B). We will clarify these points in the text.
Regarding cytoplasmic versus membrane-associated mRNAs, although we did not have a membrane marker, we relied on the brightness of the DLG-1::GFP signal to identify the cell borders (i.e., membranes) after over-exposure. This approach allowed us to discern apicobasal and apical sides for the intensity profile analyses. We will clarify this point as well in the text and, in parallel, we will try a different approach using transverse sections on top views to clarify our data.
To my eye, it seems that Figure 5 could be more faithfully interpreted to state that DGL-1 protein localization depends on the L27-SH3 domains. The Huk/Guk domains are dispensable for DLG-1 protein localization; however, through other studies, we know they are important for viability. In contrast, dlg-1 mRNA localization requires all domains of the protein (L27-Guk). It is exceptionally interesting to find a mutant condition in which the mRNA and protein localizations are uncoupled. It would be very interesting to explore in the discussion or by other means what the purpose of localized translation may be. Because, in this instance, proper mRNA localization and protein function are closely associated, it may suggest that DLG-1 needs to be translated locally to function properly.
We will rewrite the Results and Discussion to clarify our model. We agree that L27 and SH3 domains are critical, but we also detected effects of the HooK/GuK domains. We have refined our model to describe functions of the N and C termini for membrane or junctional localization.
The manuscript requires an improve materials & methods description of the quantification __procedures and statistics employed.__
We will add these points.
Minor & Major comments together - text
Summary statement: Is "adherent junction" supposed to be "adherens junction?"
Corrected.
Abstract: Sentence 1, I think they should add a caveat word to this sentence. Something like "...phenomenon that can facilitate sub-cellular protein targeting." In most instances this isn't very well characterized or known.
Corrected.
In the first paragraph, it might be good to mention that Moor et al also showed that mRNA localize to different regions to alter their level of translation (to concentrate them in high ribosome dense regions of the cell).
Added as follows: “For example, a global analysis of localized mRNAs in murine intestinal epithelia found that 30% of highly expressed transcripts were polarized and that their localization coincided with highly abundant regions in ribosomes **(Moor, 2017).”
There are some new studies of translation-dependent mRNA localization - that might be good to highlight - Li et al., Cell Reports (PMID: 33951426) 2021; Sepulveda et al., 2018 (PCM), Hirashima et al., 2018; Safieddine, et al 2021. Also, Hughes and Simmonds, 2019 reviews membrane associated mRNA localization in Drosophila. And a new review by Das et al (Nat Rev MCB) 2021 is also nice.
We will add them to the text.
Parker et al. did not show that the 3'UTR was dispensable for mRNA localization. They showed the 3'UTR was sufficient for mRNA localization.
Quoting from the paper Parker et al.: “3′UTRs of erm-1 and imb-2 were not sufficient to drive mRNA subcellular localization. Endogenous erm-1 and imb-2 mRNAs localize to the cell or nuclear peripheries, respectively, but mNeonGreen mRNA appended with erm-1 or imb-2 3′UTRs failed to recapitulate those patterns (Fig. 4A-D).” We will make this point clearer in the rewritten text.
In the second paragraph, the sentence about bean stages is missing one closing parenthesis.
Corrected.
Last paragraph: FISH is fluorescence, not fluorescent.
Corrected.
Both "subcellular" and "sub-cellular" are used.
Corrected.
Minor comments – Figures
Figure 1
o Figure 1A is confusing. It's not totally clear what the rectangles and circles signify. There are many acronyms within the figure. Which of the cell types depicted in the figure are shown here? For example, for the dorsal cells, which is the apical v. basal side?
We tried to simplify the cartoon for a general C. elegans epithelial cell. We followed schematics already shown in previous publications to maintain consistency. Acronyms and color-codes are listed in the corresponding figure legend and have been better clarified.
o Some of the colors are difficult to distinguish, particularly when printed out or for red/green colorblind readers. Is erm-1 meant to be a cytoskeletal associated or a basolateral polarity factor?
We understand the issue, but unfortunately, with 8 classes of factors, shades of gray might not solve the problem. We tried to circumvent the red-green issue changing red to dark grey. Furthermore, we added details about shapes to the figure legends. We will work to make the colors work better.
ERM-1 is a cytoskeletal-associated factor.
o The nomenclature for dlg-1 is inconsistent within "C".
Corrected.
o Please specify what the "cr" is in "cr.dlg-1:-gfp" in the legend.
Added.
Figure 2
o Can Figure 2C be quantified in a similar manner to 2A/2B?
Currently our script cannot do that, but we will try to optimize it to be able to quantify this type of images.
o 2B - please jitter the dots to better visualize them when they land on top of one another
Yes, we will.
o Please include a negative control example, a transcript that is not peripherally localized for comparison.
Yes, we will.
o There is no place in the text of the document where Fig 2C is referenced
Corrected (it was wrongly referred to as “2B”).
o I can't see any discernable ajm-1 localization in Fig 2A.
We added some arrowheads to point at specific examples and increased the intensities of the corresponding smFISH signal for better visualization.
o I can't see any dlg-1 pharyngeal localization in Fig2C.
We added some arrowheads to point at specific examples and increased the intensities of the corresponding smFISH signal for better visualization.
o More details on how the quantification was performed would be welcome. Particularly, in 2B, what is the distance from the membrane in which transcripts were called as membrane-associated? What statistics were used to test differences between groups?
We will add a full description of the script used as well as the statistic details.
Figure 3
o Totally optional but might be nice: can you make a better attempt to approximate the scale of the cartoon depiction?
The UTRs, especially the 5’ one, are much smaller than the dlg-1 gene sequence. A proper scaling of the cartoon to the actual sequences, would draw the attention away from the main subjects of this figure, the UTRs. Nevertheless, we made sure it is clear in the corresponding figure legend that the cartoon is not in scale: “The schematics are not in scale with the actual size of the corresponding sequences. UTR lengths: dlg-1 5’UTR: 61 nucleotides; sax-7 5’UTR: 63 nucleotides; dlg-1 3’UTR: 815 nucleotides; unc-54 3’UTR: 280 nucleotides.”
o The GFP as an asterisk illustration may be confusing for some readers. Could you add another rectangular box to depict the gfp coding sequence?
Corrected.
o This microscopy is beautiful!
Thanks Reviewer #2!
o Were introns removed? Is the endogenous copy still present?
All the transgenes were analyzed in a wild-type background, therefore, yes, the endogenous copy was still present. All the transgenes possessed introns. We will change the corresponding text as follows: “To test whether the localization of one of the identified localized mRNAs, dlg-1, relied on zip codes, we generated extrachromosomal transgenic lines carrying a dlg-1 gene whose sequence was fused to an in-frame GFP and to exogenous UTRs.”. In the figure “dlg-1 ORF” has been replaced with “dlg-1 gene”.
o The wording in the legend "CRISPR or transgenic" may be confusing as Cas9 genome editing is still a form of transgenesis.
We added “extrachromosomal” to clarify the nature of the mRNA.
o The authors state that the 5'-3'UTR construct produces perinuclear dlg-1 transcripts but in the absence of DAPI imaging, it's not clear that this is the case.
We could not find such a statement, but we tried to clarify the localization of these mRNAs in the text: “The mRNA localization patterns of the two UTR reporters were compared to the localization of dlg-1 transcripts from the CRISPR line (“wild-type”, Fig. 3A; Heppert et al., 2018), described in Fig. 2. Both reporter strains showed enrichment at the CeAJ and localization dynamics of their transcripts that were comparable to the wild-type cr.dlg-1 (Fig. 3B). These results indicate that the UTR sequences of dlg-1** mRNA are not required for its localization.”
o Which probe set was used? The gfp probe?
Yes, please see the main text: “Given that the transgenic constructs were expressed in a wild-type background, smFISH experiments were conducted with probes against GFP RNA sequences to focus on the transgenic dlg-1::GFP mRNAs (cr.dlg-1 and tg.dlg-1).”
o Here, sax-7 is used as a uniform control, but sax-7 is claimed in Fig S1B-D as being perinuclear. This is a bit confusing.
sax-7 mRNA localizes perinuclearly in sporadic instances (Fig S1C), but it is predominantly scattered throughout the cytoplasm (i.e., unlocalized). It presumably localizes perinuclearly in a translation-dependent manner as sax-7 codes for a transmembrane protein that would be targeted to the ER. We have described this ER-type of localization in the introduction and reiterated it partially in the first paragraph of the results. sax-7 UTRs are therefore presumably not responsible for any subcellular localization, which would instead rely on a signal sequence. We will better clarify this point in the main text.
Figure 4
o Excellent results! Really nice!
Thanks Reviewer #2!
o Fig 4A. The GFP depicted as a circle is strange.
We changed it into a rectangle.
o Fig 4A. Can you include the gene/protein name for easy skimming?
Added.
o Fig 4B. the color here is too faint and it is unclear what is being depicted. Overall, this part of the figure could be improved.
We are optimizing the coloring and simplifying the schematics.
o Were the introns removed?
No, the introns were maintained in this and in all our transgenic lines. We described our transgenic lines in the materials and methods section (now with more detail). What we depict in the scheme (Fig. 4A) is the mature RNA (now specified in the figure), therefore no introns depicted. We will also specify this in the main text.
Figure 5
o Fig 5A. can you add the gene/protein name
Added.
o Fig 5B. Can you make the example apicobasal (non-apical) mRNA more distinctive? If it had its own peak in the lower trace, the reader would more clearly understand that this mRNA will be excluded from apical measurements whereas it will be included in apicobasal measurements.
We actually wanted to show this specific example: a cytoplasmic mRNA and a junctional mRNA may seem close from the apicobasal analysis (partially overlapping peaks that Reviewer #2 mentioned). With the apical analysis, instead, we can show that these mRNAs are actually not close, and they belong to two different compartments (cytoplasm and junction). We would therefore like to keep the current scheme, while better clarifying this point in the corresponding figure legend.
o D' - I' The grey font is too light.
Noted. We will change it.
o D' - I' The inconsistent y-axis scaling makes it difficult to compare across these samples. Can you set them to the same maximum number?
The values are indeed quite different. We tried to use the same scale, but this would make some of the data unappreciable. The idea was to evaluate, within each graph, how mRNA and protein are localized relative to the junctional marker. We will make this clearer in the text.
o D' - I' The x-axis labels are formatted incorrectly
Corrected.
o The practice of masking out the nucleus appears to remove potentially important mRNAs that are not nuclear localized. This could really impact the findings and interpretation. Instead, consider an automated DAPI mask.
The masking on the images is not the same used for the analysis: in the images, a shaded circle has been drawn on the DNA channel and moved onto its corresponding location in the other channels or merges. For the analysis, the DNA signal has been specifically removed in the channel with the smFISH signal. Given that the analysis has been performed on maximum intensity projections of 3 Z-stacks, we believe we did not remove any non-nuclear mRNA. We will clarify this point in Materials and methods.
o I can't see what the authors are calling membrane diffuse versus cytoplasmic. This is making it hard for me to see their "two step" pathway to localization.
We will add in Fig. 5B-C an example of a membrane localized mRNA. Furthermore, we will add transverse sections of membrane and cytoplasm to make the date clearer to the reader.
o Can more details of the quantification be included? How were Z-sections selected, chosen for inclusion? Which Z-sections and how many were selected?
We will add the details to Materials and methods.
o Also, why do these measurements focus on what I think are the seam cells when Lockwood et al., 2008 show the entire epithelium that is much easier to see?
We are focusing on the seam cells at the bean stage as these are the cells and the embryonic stage where we see the highest localization of dlg-1 mRNA in the wild-type.
o Please name these constructs to correlate the text more explicitly to the figures.
Added.
o How many embryos were analyzed for each trace? How many embryos showed consistent patterns?
We will add the details of the analysis to Materials and methods.
o Why were these cells used for study here? Lockwood et al., 2008 use a larger field of epithelial cells for visualization.
As stated before: we are focusing on the seam cells at the bean stage as these are the cells and the embryonic stage where we see the highest localization of dlg-1 mRNA in the wild-type.
Figure 6
There are major discrepancies between what this figure is depicting graphically and what is described in the text. Again, I'm not comfortable making the "two step" claims this figure purports given the data shared in Figure 5.
We are planning to re-write the last part of the results to better clarify our two-step model. A two-step model had been previously suggested in McMahon et al., 2001, where they could show that DLG-1 and AJM-1 (referred to in that publication as JAM-1) are initially localized laterally and only later in development are then enriched apically. Our data agree with McMahon very well, so we used the earlier study as a start. We will cite and explain this paper in greater depth during the rewriting.
**Minor comments - Tables & Supplemental Figures**
Table 1
I think this table could be improved to more clearly illustrate which mRNAs were tested and what their mRNA localization patterns were (for example, gene name identifiers included, etc). Could the information that is depicted by gray shading instead be added as its own column? For example, have a column for "Observed mRNA localization"
We modified Table 1 based on these and the other reviewers’ comments.
Can you add distinct column names for the two columns that are labeled as "protein localization - group"
We modified Table 1 based on these and the other reviewers’ comments.
Can you also add which of these components are part of ASI v. ASII (as described in the introduction?)
A new table has been added with the factors belonging to the two adhesion systems (same color code as in Table 1).
Supplemental Figure 1
It is hard to see that some of these spots are perinuclear. More information (membrane marker, 3D rendering, improved metrics) is required to support this claim.
We are not trying to make a big statement out of these data as perinuclear localization for mRNAs coding for transmembrane/secreted proteins is well known. The aim of our study was to describe transcript localized at or in the proximity of the junction. We thought it was worth mentioning these examples of perinuclearly localized mRNAs (hmr-1, sax-7, and eat-20) for two reasons: scientific correctness – show accessory results that might be interesting for other scientists – and use as positive controls for our smFISH survey – these mRNAs were expected to have a somewhat perinuclear localization for the reasons mentioned above.
What do these images look like over the entire embryo, not just in the zoomed in section?
We added a column with the zoom-out embryos.
sax-7 localization in S4 looks similar but a different localization claim is made.
sax-7 mRNA can localize perinuclearly in sporadic instances (Fig S1C), but is predominantly scattered throughout the cytoplasm (i.e., unlocalized). It presumably localizes perinuclearly in a translation-dependent manner as sax-7 codes for a transmembrane protein that would be targeted to the ER. We have described this ER-type of localization in the introduction and reiterated it partially in the first paragraph of the results. sax-7 UTRs are therefore presumably not responsible for any subcellular localization, which would instead rely on a signal sequence. We will better clarify this point in the main text.
Supplemental Figure 2
Before adherens junctions even exist dlg-1 go to the membrane - this is really neat!
Thanks Reviewer #2!
Supplemental Figure 3
Technical question: If either 5 or 3 stack images are used, how does this work? Do they have different z-spacings? Or do they do 5-stack images represent a wider Z-space?
This is the sentence under question: “Maximum intensity projections of 5 (1.08 µm) (A) and 3 (0.54 µm) (B) Z-stacks”. The space between each Z-stack image is constant in all our imaging and its value is 270 nm. When we consider 5 planes, the distance from the 1st to the 5th is 4 x 270 nm = 1.08 µm, whereas for 3 planes will be 2 x 270 nm = 0.54 µm.
Supplemental Figure 4
Line #2 retains translation and keeps mRNA localization.
Totally optional, but consider showing both lines in the main figure to illustrate the two possibilities.
Noted.
Materials and methods - how did they created the ATG mutations? Is it an array? - why does one translate, and one doesn't?
We will clarify this point in Materials and methods: “dlg-1 deletion constructs ΔATG (SM2664 and SM2663) and ΔL27-PDZs (SM2641) were generated by overlap extension PCR using pML902 as a template.”.
We will perform a Western blot to clarify Reviewer #2’s last point. Currently we do not know what peptide is translated, but the comparison with our full-length control will probably shed some light on the issue.
Reviewer #3
Major comments
The smFISH results are striking and implications exciting. The conclusions made from the smFISH results reported in all Figures will be strengthened considerably by quantifying the mRNA localized to the defined specific subcellular regions. At the very least, localization to the cytoplasm versus the plasma membrane should be determined as performed in Figure 2B, but quantifying finer localization will enhance the conclusions made about regional localization (e.g. CeAJ versus plasma membrane mRNA localization in Figure 5). Inclusion of a non-localizing control in Figures 1-4 will enable statistical comparisons between mRNA localizing and non-localizing groups.
We will add more quantitation, statistics, and negative controls.
The script used for smFISH quantitation should be included in the methods or published in an accessible forum (Github, etc). Criteria for mRNA "dot" calling should be defined in the methods. All raw smFISH counts should also be reported.
We will add the full description of the script in Materials and methods, and we will provide the raw data in an additional supplementary table.
Figure 2: What is the localizing ratio of a non-localizing control mRNA (e.g. jac-1)? Including an unlocalized control with quantitation would strengthen the localization arguments presented.
Yes, we will add quantitation for an unlocalized mRNA.
Figure 5: Quantifying colocalization of mRNA and protein (+/- AJM-1) will strengthen the arguments made about mRNA/protein localization.
Yes, we will quantify Fig. S5 to have a full picture of the cells (the images in Fig. 5 represent only a portion of the cell).
Discussion of the CeAJ mRNA localization mechanism is warranted. Do the authors speculate that the newly translated protein drives localization during translation, similar in concept to SRP-mediated localization to the ER, or ribosome association is a trigger to permit a secondary factor to drive mRNA localization, or another model?
Unfortunately, this is hard to say at the moment as we do not have any data regarding where translation actually occurs. We will add a conjecture to the Discussion.
Minor comments
Please complete the following sentence: "We identified transcripts enriched at the CeAJ in a stage- and cell type-specific."
Corrected.
It would be helpful to provide reference(s) for the protein localization summary in Table 1.
Added.
Figure 2B: Did dlg-1 and ajm-1 localize at similar ratios? Appropriate statistics comparing the different ratios may be informative.
We will modify the graph (following Reviewer #2’s suggestion) and add the requested details.
Figure 2: In the paragraph that begins, "Morphogenesis of the digestive track," the text should refer to Figure 2C? If not, the text requires further clarification.
Corrected.
Figure 2: Reporting the smFISH localizing ratios of 8E and 16E will be informative.
We will add the information.
Please include citations when summarizing the nonsense-mediated decay NMD mechanism and AJM-1 identifying the CeAJ.
Added.
The sentence, "Embryos from our second __Δ__ATG transgenic line displayed a little GFP protein and some dlg-1::gfp mRNA," should refer to Figure S4.
Added.
An immunoblot of this reporter versus wild type may be informative regarding the approximate position of putative alternative start codon.
We will perform a Western blot to verify the size of the protein product produced.
Figure 5: N's and repetitions performed should be included for localization experiments.
Yes, we will add them here and in all the other quantifications we will add to the manuscript.
Please clarify that the "the mechanism of UTR-independent targeting is unknown in any species" refers to dlg-1 mRNA localization.
Added.
"Our findings suggest..." discussion paragraph should reference Figure 6.
Added.