Dynamics of bicoid mRNA localisation and translation dictate morphogen gradient formation

  1. T. Athilingam
  2. E.L. Wilby
  3. P. Bensidoun
  4. A. Trullo
  5. M. Verbrugghe
  6. X. Shi
  7. M. Lagha
  8. T.E. Saunders
  9. T.T. Weil

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Abstract

The transcription factor Bicoid (Bcd) protein guides early Drosophila patterning and is the best-characterised morphogen. The source of the morphogen, bcd mRNA, is maternally deposited during oogenesis and localised to the anterior pole of the mature oocyte. While the spatiotemporal interpretation of the Bcd morphogen gradient has been intensely studied, when and where Bcd protein is produced and how this protein gradient is dynamically shaped remains contentious. Here, we use the SunTag reporter system to quantitatively examine the spatiotemporal profile of bcd mRNA translation in vivo . We show that association with Processing bodies (P bodies) in mature oocytes prevent premature bcd mRNA translation. Following egg activation, bcd mRNA dissociates from P bodies and translation is observed at the anterior pole. Translation remains restricted to the anterior domain throughout early development, even after nuclear migration in the syncytial blastoderm. At cellularisation, translation ceases and the remaining bcd mRNA associates with reformed P bodies, which appear to block any further translation. We use these observations to create a new modified source-diffusion-degradation model of Bcd gradient formation that has spatiotemporally varying production. Overall, our study reveals that bcd mRNA translation is tightly controlled in space and time during oogenesis and early embryogenesis.

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    Reply to the reviewers

    __Reply to the Reviewers __

    We thank the Reviewers for their positive assessment and recognition of the paper achievements. The insightful comments will strengthen the data and manuscript.

    Referee #1*

    Minor comments

    1. Fig 1B - add arrows showing mRNAs being translated or not (the latter mentioned in line 113 is not so easy to see). We have magnified the inset of the colocalisation in the right column; we added arrows and arrowheads to differentiate colocalised and non-colocalised bcd with translating SunTag.
    • Fig 2A - add a sentence explaining why 1,6HD, 2,5HD and NaCl disrupt P bodies.

    We have added the information on the use of 1,6HD, 2,5HD, and NaCl to disrupt P-bodies as below. Revised line 158: “To further show that bcd storage in P bodies is required for translational repression, we treated mature eggs with chemicals known to disrupt RNP granule integrity (31, 37, 69-72). Previous work has shown that the physical properties of P bodies in mature Drosophila oocytes can be shifted from an arrested to a more liquid-like state by addition of the aliphatic alcohol hexanediol (HD) (Sankaranarayanan et al., 2021, Ribbeck and Görlich, 2002; Kroschwald et al., 2017). While 1,6 HD has been widely used to probe the physical state of phase-separated condensates both in vivo and in vitro (Alberti et al., 2019; McSwiggen et al., 2019; Gao et al., 2022), in some cells it appears to have unwanted cellular consequences (Ulianov et al., 2021). These include a potentially lethal cellular consequences that may indirectly affect the ability of condensates to form (Kroschwald et al., 2017) and wider cellular implications thought to alter the activity of kinases (Düster et al., 2021). While we did not observe any noticeable cellular issues in mature Drosophila oocytes with 1,6 HD, we also used 2,5 HD, known to be less problematic in most tissues (Ulianov et al., 2021) and the monovalent salt sodium chloride (NaCl), which changes electrostatic interactions (Sankaranarayanan et al., 2021).”

    *Fig 4C - explain in the legend what the white lines drawn over the image represent. And why is there such an obvious distinction in the staining where suddenly the DAPI is much more evident (is the image from tile scans)?

    Figure 4C is the tile scan image of a n.c.10 embryo and the white line classified the image into four quadrants. We used this image to quantify the extent of bcd (magenta) colocalisation to SunTag (green) in the anterior and posterior domains of the embryo in the bar graph shown in panel C’. There is a formatting error in the image. We will correct this in the revised version. We will also include the details of white lines in the legends. Finally, based on further reviewer comments, in the revised version this data is shifted to the supplementary information.

    • Line 215 - 'We did not see any significant differences in the translation of bcd based on their position, however, there appears an enhanced translation of bcd localised basally to the nuclei (Figure S5).' Since the difference is not significant, I do not think the authors should conclude that translation is enhanced basally.

    We agree with the reviewer. In this preliminary revision we have changed this statement to: “We did not see any differences in the translation of bcd based on their position with respect to the nuclei position (Figure S5)” (revised line 238-239).

    *Line 218: 'The interphase nuclei and their subsequent mitotic divisions appeared to displace bcd towards the apical surface (Figure S6B).' Greater explanation is needed in the legend to Fig S6B to support this statement as the data just seem to show a nuclear division - I would have thought an apical-basal view is needed to conclude this.

    We have rearranged this figure and shown in clarity the apical-basal view of the blastoderm nuclei and the displacement of bcd from the surface of the blastoderm in Figure S8.

    New Figure S8: n.c.8 - pre-cortical migration; n.c.12,14- post cortical migration; Mitosis stages of n.c.9-10. The cortical interphase nuclei at n.c. 12,14 displaces bcd. The nuclear area (DAPI, cyan) does not show any bcd particles (magenta) indicated by blue stars. The mitotic nuclei (yellow arrowheads, yellow stars) displace bcd along the plane of nuclear division (doubled headed yellow arrows).

    Fig 5B - the authors compare Bcd protein distribution across developmental time. However, in the early time points cytoplasmic Bcd is measured (presumably as it does not appear nuclear until nc8 onwards) and compare the distribution to nuclear Bcd intensities from nc9 onwards. Is most/all of the Bcd protein nuclear localised form nc9 to validate the nuclear quantitation? Does the distribution look the same if total Bcd protein is measured per volume rather than just the nuclear signal? Are the authors assuming a constant fast rate of nuclear import?

    From n.c.8 onwards, the Bcd signal in interphase nuclei builds up, with the nuclear intensity becoming very high compared to cytoplasmic Bcd. However, we do see significant Bcd signal in the cytoplasm (*i.e., *above background). In earlier work, gradients of the nuclear Bcd and nuclear-import mutant Bcd overlapped closely (Figure 1B, Grimm et al., 2010). This essentially suggests the nuclear Bcd gradient reflects the corresponding gradient of cytoplasmic Bcd. Further, the nuclear import of Bcd occurs rapidly after photobleaching (Gregor et al., 2007). Based on these observations, and our own measurements, prior to n.c. 9, the cytoplasmic gradient is likely a good approximation of the overall shape, whereas post n.c. 9 the Bcd signal is largely nuclear localised. Further, the overall profile is not dependent on the nuclear volume.

    • Line 259 - 'We then asked if considering the spatiotemporal pattern of bcd translation' - the authors should clarify what new information was included in the model. Similarly in line 286, 'By including more realistic bcd mRNA translation' - what does this actually mean? In line 346, 'We see that the original SDD model .... was too simple.' It would be nice to compare the outputs from the original vs modified SDD models to support the statement that the original model was too simple.

    We will improve the linking of the results to the model. The important point is that when and where Bcd production occurs is more faithfully used, compared with previous approximations. By including more realistic production domains, we can replicate the observed Bcd gradient within the SDD paradigm without resorting to more complex models.

    Fig S1A - clarify what the difference is between the 2 +HD panels shown.__ __

    The two +HD panels at stage 14 indicate that upon the addition of HD, there are no particles in 70% of the embryos, and 30% show reduced particles. We will add this information to the figure legend.

    • Fig S2E - the graph axis label/legend says it is intensity/molecule. Since intensity/molecule is higher in the anterior for bcd RNAs, is this because there are clumps of mRNAs (in which case it's actually intensity/puncta)?

    The density of mRNA is very high in the anterior pole; there is a chance that more than one *bcd *particle is within the imaged puncta (due to optical resolution limitations). We will change the y-axis to average intensity per molecule to average intensity per puncta.

    • Fig S4 - I think this line is included in error: '(B) The line plots of bcd spread on the Dorsal vs. Ventral surfaces.'*

    Yes, we will correct this in the revision.

    • In B, D, E - is the plot depth from the dorsal surface? I would have preferred to see actual mRNA numbers rather than normalised mRNAs. In Fig S4D moderate, from 10um onwards there are virtually no mRNA counts based on the normalised value, but what is the actual number? The equivalent % translated data in Fig S4E look noisy so I wonder if this is due to there being a tiny mRNA number. The same is true for Figs S4D, E 10um+ in the low region.*

    Beyond 10um from the dorsal surface, the number of bcdsun10 counts is very low. It becomes negligible at the moderate and low domains. We will attach the actual counts of mRNA in all these domains as a supplementary table in the revised version.

    General assessment* Strengths are: 1) the data are of high quality; 2) the study advances the field by directly visualising Bcd mRNA translation during early Drosophila development; 3) the data showing re-localisation of bcd mRNAs to P bodies nc14 provides new mechanistic insight into its degradation; 4) a new SDD model for Bcd gradient formation is presented. Limitations of the study are: 1) there was already strong evidence (but no direct demonstration) that bcd mRNA translation was associated with release from P bodies at egg activation; 2) it is not totally clear to me how exactly the modified SDD model varies from the original one both in terms of parameters included and model output.*

    This is the first direct demonstration of the translation of bcd mRNA released as a single mRNA from P bodies. Previously, we have shown that P bodies disruption releases single bcd from the condensates (31). We have captured a comprehensive understanding of the status of individual bcd translation events, from their release from P bodies at the end of oocyte maturation until the end of blastoderm formation.

    The underlying SDD model – that of localised production, diffusion, and degradation – is still the same (up to spatially varying diffusion). Yet the model as originally formulated did not fit all aspects of the data, especially with regards to the system dynamics. Here, we demonstrate that by including more accurate approximations of when and where Bcd is produced, we can explain the formation of the Bcd morphogen gradient without recourse to any further mechanism.

    Referee #2

    1. Line 114: The authors claim to have validated the SunTag using a fluorescent reporter, but do not show any data. Ref 60 is a general reference to the SunTag, and not the Bcd results in this paper. Perhaps place their data into a supplemental figure or movie? To show the validation of our *bcdSun32 *line, we have composed a new Figure S1 that shows the translating *bcdSun32 *(magenta) colocalising to the ScFV-mSGFP2 (green). Yellow arrowheads in the zoom (right panel) points to the translating *bcdSun32 *(magenta) and red arrowheads points to the untranslated bcdSun32. In addition, we have also shown the validation of bcdSun32 with the anti-GCN4 staining in the main Figure 1B.

    Further, we have dedicated supplementary Figure S3 (previously Figure S2) for the validation of our bcdSun10 construct. Briefly, bcdSun10 is inserted into att40 site of chr.2. We did a rescue experiment, where bcdSun10 rescued the lethality of homozygous bcdE1 null mutant. We then performed a colocalisation experiment using smFISH, where we demonstrated that almost all bcd in the anterior pole are of type bcdSun10. We targeted specific fluorescent FISH probes against 10xSunTag sequence (magenta, Figure S2A) and bcd coding sequence (magenta, Figure S2A). Upon colocalisation, we found ~90% of the mRNA are of bcdSun10 type. The remaining 10% could likely be contributed by the noise level (Figure S2B). We will make sure these points are clear in the revised manuscript.

    Line 128 and Fig. 1E: The claim that bcd becomes dispersed is difficult to verify by looking at the image. The language could also be more precise. What does it mean to lose tight association? Perhaps the authors could quantify the distribution, and summarize it by a length scale parameter? This is one of the main claims of the paper (cf. Line 23 of the abstract) but it is described vaguely and tersely here.

    We have changed the text from, “We also confirmed that bcd becomes dispersed, losing its tight association with the anterior cortex (Figure 1E) (31)” to, “We also confirmed that bcd is released from the anterior cortex at egg activation (Figure 1E) (31, 21).” (Revised line 131).

    The release of bcd mRNA at egg activation was first shown in 2008 (Ref 21, Figure 4, D-E) and again in 2021 (Ref 31, Figure 7 B and E). The main point in line 127-128, “P bodies disassembled and *bcd *was no longer colocalised with P bodies” and the novel aspect of line 23 is “translation observed”. The distribution of bcd mRNA after egg activation was not the point of this section. We have improved the writing in the revision to make this clearer.

    Line 146, Fig. 1G: This is a really important figure in the paper, but it is confusing because it seems the authors use the word "translation," when they mean "presence of Bcd protein." In other places in the paper, the authors give the impression that "bcd translation" means translation in progress (assayed by the colocalization of GCN4 and bcd mRNA). However, in Fig. 1G, the focus is only on GCN4. Detecting Bcd protein only at the anterior does not mean that translation happens only at the anterior (e.g., diffusion or spatially-restricted degradation could be in play).

    In Figure 1G, we have shown only the “translated” Bcd by staining with a-GCN4. We have changed line 146 from, “Consistent with previous findings, we only observed bcd translation at the anterior of the activated egg and early embryo (Figure 1G-H) (3, 68)” to, “Consistent with previous findings, we only observed the presence of Bcd protein at the anterior of the activated egg and early embryo (Figure 1G-H) (3, 68). (Revised line 151-153). We will use “translating bcd” or “bcd in translation” where we show colocalisation of bcd with BcdSun10 or BcdSun32 elsewhere in the manuscript.

    We did not mean to claim that translation occurred only in the anterior pole. We show that the abundance of bcd is very high in the anterior pole (in agreement with previous work) and that this is where the majority of observed translation events took place. Indeed, we have also shown that posteriorly localised mRNAs have the same BcdSun10 intensity per bcd puncta from the posterior pole (Figure 3B & 4C’ and Figure S2 E), but these are much fewer in number.

    *It would also be helpful to show a plot with quantification of Bcd detection (or translation) on the y-axis and a continuous AP coordinate on the x-axis, instead of just two points (anterior and posterior poles, the latter of which is uninteresting because observing no Bcd at the posterior pole is expected).

    In Figure 1G,H, our aim was to test whether release from P bodies allowed for bcd mRNA to be translated. We used the presence of Bcd protein at the anterior domain of the oocytes to show this. The posterior pole was included as an internal control. To show the spatial distribution of bcd mRNA and its translation, we used early blastoderm (Figure 3, Figure S4).

    Another issue with Fig. 1G is that the A and P panels presumably have different brightness and contrast. If not, just from looking at the A and P panels, the conclusion would be that Bcd protein is diffuse (and abundant) in the posterior and concentrated into puncta in the anterior. The authors should either make the brightness and contrast consistent or state that the P panel had a much higher brightness than the A panel.

    We agree with this shortcoming. We have now added the following to Figure 1 legend to clarify this observation. “G: Representative fixed 10 µm Z-stack images (from 10 samples) showing BcdSun32 protein (anti-GCN4) is only present at the anterior of an in vitro activated egg or early embryo 30-minute post fertilization. BcdSun32 protein is not detected in these samples at the posterior pole (image contrast increased to highlight the lack of distinct particles at the posterior). BcdSun32 protein is also not detected at the anterior or posterior of a mature oocyte or an in vitro activated egg incubated with NS8953 (images have the contrast increased to highlight the lack of distinct particles). Scale bar: 20 mm; zoom 2 mm.” (Revised line 623).

    • Line 176: This section is very confusing, because at this point the authors already addressed the spatial localization of translation in Fig. 1G,H (see my above comment). However, here it seems the authors have switched the definition of translation back to "translation in progress." Therefore, the confusion here could be eliminated by addressing the above point.*

    In the revised version, we will use Bcd protein when shown with anti-GCN4 staining. We will use “translating bcd” or “bcd in translation” where we show colocalisation of bcd with a-GCN4 (BcdSun10 or BcdSun32). We will change this in the corresponding text.

    Line 185: The sentence here is seemingly contradictory: "most...within 100 microns" implies that at least some are beyond 100 microns, while the sentence ends with "[none]...more than 100 microns." The language could perhaps be altered to be less vague/contradictory.

    We will clarify this in the revised version. There are few particles visible beyond 100 um. In the lower panel of Figure 3B, the posterior domain shows few particles. However, their actual number compared to bcd counts within the 100 um is negligible (Figure3C). Nonetheless, the few bcd particles we observe do seem to be under translation (quantified in Figure 4C’ and Figure S2E).

    • Line 204: It would be really nice to have quantification of the translation events, such as curves of rate of translation as a function of a continuous AP coordinate, and a curve for each nc.*__ __

    In the revised version we will provide the results quantifying the translation events across the anterior- posterior axis. This will provide a clarity to the presence of bcd and their translation in the posterior domain with time.

    Our colocalisation analysis is semi-automated. It includes an automated counting of the individual bcd particle counts and a manual judgement of the colocalised BcdSun10 protein (distinct spots, above noise) to bcd particles (Figure S3D). The bcd particle counts ran into thousands in each cyan square box (measuring 50um radius and ~ 20um deep from the dorsal surface). We selected three such boxes covering 150um (continuously) from the anterior pole across A-P axis and 20um deep of the flattened embryo mounts across D-V axis (Figure 3A-C, Figure S4). We have also scanned scarce particles in the posterior; however,* bcd* counts are very low compared to the anterior. Further, in Figure 4 we have repeated the same technique to measure translation of bcd particles in embryos at different nuclear cycles.

    We have also shown continuous intensity measurements of bcd particles with their respective BcdSun10 gradient in Figure 5 across the A-P axis at different nuclear cycles. Here, we know BcdSun10 intensity is not only from the “translating” bcd (colocalised BcdSun10 to bcd particles) but also from the translated BcdSun10 freely diffusing (non-colocalised BcdSun10 to bcd particles). As asked by the reviewer, in the revised version we will add bcd counts and their translation status from anterior to posterior axis for each of the nuclear cycles.

    In our future work, we planned to generate MS2 tagged bcdSun10 to measure the rates of translation in live across all nuclear cycles.

    *Line 209 and Fig 4C: The authors use the terms "intensity of translation events" or "translation intensity" without clearly defining them. From the figure (specifically from the y-axis label), it looks like the authors are quantifying the intensity per molecule (which is not clearly the same thing as "translation intensity"), but it would be nice if that were stated explicitly.

    In the relevant result section, we have changed the results text to “the intensity of translation events” for explaining the results of Figure 4C’.

    • In addition, the authors again quantify only two points. This is a continuously frustrating part of the manuscript, which applies to nearly all figures where the authors looked only at two points in space. At a typical sample size of N = 3, it seems well within time constraints to image at multiple points along the AP axis.*__ __

    In addition to the quantification shown at the anterior and posterior locations of the embryo in the Figure 3 and 4, we will show in the revised version, the quantification of translation events across all locations from the anterior to the posterior. We will use three embryos for each nuclear cycle from n.c.1 to 14.

    • Furthermore, it sounds like the authors are saying the "translation intensity" is the same in anterior and the posterior, which is counterintuitive. The expectation is that translation would be undetectable at the posterior end, in part because bcd mRNA would not be present. (Note that this expectation is even acknowledged by the authors on Line 185, which I comment on above, and also on Line 197). There should also be very low levels of Bcd protein (possibly undetectable) at the posterior pole. As such, the authors should explain how they think their claim of the same "translation intensities" in the anterior vs posterior fits into the bigger picture of what we know about Bcd and what they have already stated in the manuscript. They should also explain how they observed enough molecules to quantify at the posterior end. The authors should also disclose how many points are in each box in the boxplot. For example, the sample size is N = 3 embryos. In just three embryos, how many bcd/GCN4 colocalizations did the authors observe at the posterior end of the embryo?*

    In n.c.4 in Figure3, we saw few bcd particles in the posterior. However, at n.c.10 in Figure 4C’ the number of posterior bcd particles are higher than at the early stages. We have quantified them in Figure 4C’. We will clarify this from the new set of quantification we are undertaking now to quantify translation across the A-P axis in the revision.

    Finally, we will also provide the number of bcd particle counts and their colocalisation with a-GCN4 as a supplementary table.

    • Line 215: The sentence that starts on this line seems self-contradictory: I cannot tell whether or not there is a difference in translation based on position.

    We have not observed any difference in the translation of bcd particles depending on the position along the Z-axis. We will edit this in our revised version.

    • Line 229: Long-ranged is a relative term. From the graph, one could state there is some spatial extent to the mRNA gradient, so it is unclear what the authors mean when they say it is not "long-ranged." Could the mRNA gradient be quantified, such as with a spatial length scale? This would provide more information for readers to make their own conclusions about whether it is long-ranged.*

    We have quantified the *bcd *mRNA gradient for each n.c. (Figure 5B-C); absolute bcd intensities in Figure 5B, left panel and the normalised intensities in Figure 5C. The length of the mRNA spread appears constant with the half-length maximum of ~75um across all nuclear cycles. Our conclusion of a long ranged Bcd gradient is based on the comparisons of the half-length maximum measurements of bcd particles and BcdSun10 (Figure 5D).

    *Line 230: When the authors claim the Bcd gradient is steeper earlier, a quantification of the spatial extent (exponential decay length scale) would be appropriate. Indeed, lambda as a function of time would be beneficial. It should also be placed in context of earlier papers that claim the spatial length scale is constant.

    We will show this effectively from the live movies of bcdSun10/nanos-scFv-sGFP2 in the revised version.

    • Lines 235-236: The two sentences that start on these two lines are vague and seemingly contradictory. The first sentence says there is a spatial shift, but the second sentence sounds like it is saying there is no spatial change. The language could be more precise to explain the conclusions.

    We agree with the reviewer. We will edit this in revision.

    Minor comments

      • Line 81: Probably meant "evolutionarily conserved"

    Yes, we have changed, “P bodies are an evolutionarily cytoplasmic RNP granule” to, “P bodies are an evolutionarily conserved cytoplasmic RNP granule.”(Revised line 84-85).

    *Figure 1 legend: part B says "from 15 samples" but also says N = 20. Which is it, or do these numbers refer to different things?

    We have edited this from, “early embryo (from 15 samples)” to, “early embryo (from 20 samples)”. (Revised line 602).

    • Line 217: migration of what?

    Edited to “cortical nuclear migration”.

    • Line 228: "early embryo" is vague. The authors should give specific time points or nuclear cycle numbers.*

    Edited to “nuclear cycles 1-8”.

    • Line 301: Other locations in the paper say 75 microns or 100 microns.

    We will make the changes. It is 100 um.

    • Fig. 5: all images should be oriented such that the dorsal midline is on the upper half of the embryo/image.

    We will flip the image to match.

    • Fig. 5B: There are light tan and/or light orange curves (behind the bold curves) that are not explained.

    It is the standard deviation. This will be explained.

    • Fig. 5C: the plot says "normalized" but nowhere do the authors describe what the curves are normalized to. There is also no explanation for what the broad areas of light color correspond to.*__ __

    Normalised to the bcd intensity maxima. This will be explained.

    Significance

    The results, if upheld, are highly significant, as they are foundational measurements addressing a longstanding question of how morphogen gradients are formed, using Bcd (the foundational morphogen gradient) as a model. They also address fundamental questions in genetics and molecular biology: namely, control of mRNA distribution and translation.__ __

    We thank Reviewer 2 for highlighting the importance of our work in the field. We are confident that we address the issues raised by Reviewer 2 with the new set of quantifications we are currently working on.

    Referee #3

      • It is not evident from the main results and methods text that the new SDD model incorporates the phenomenon reported in figure 4B. From my reading, the parameter beta accounts for the Bcd translation rate, which according to figure 7B(ii) effectively switches from off to on around fertilization and thereafter remains constant. Figure 4B shows that the fraction of bcd mRNA engaged in translation decreases beginning around NC12/13, and this is one of the more powerful results that comes from monitoring translation in addition to RNA localization/abundance/stability. My expectation based on figure 4B would be that parameter beta should decrease over time beginning around 90-100 minutes and approach zero by ~150 minutes. This rate could be fit to the experimental data that yields figure 4B. The modeling should be repeated while including this information.* This is a good observation. Currently, the reduced rate of *bcd *translation is modelled by incorporating an increased rate of *bcd *mRNA degradation. Of course, this could also be reduced by a change in the rate of translation directly. As stated already, the beta parameter is the least well characterised. In the revision, we will include a model where beta changes but not the mRNA degradation rate. We will improve the discussion to make this point clearer.
    • The presentation of the SDD model should be expanded to address how well the characteristic decay length fits A) measured Bcd protein distributions, B) measured at different nuclear cycles. This would strengthen the claim that the new SDD model better captures gradient dynamics given the addition of translation and RNA distribution. These experimental data already exist as reported in Figure 5. In the current Figure 7, panels D and D' add little to the story and could be moved to a supplement if the authors want to include it (in any case, please fix the typo on the time axis of fig 7D' to read "hours"). The model per cell cycle and the comparison of experimental and modeled decay lengths could replace current D and D'.*

    Originally, we kept discussion of the SDD model only to core points. It is clear from all Reviewers that expanding this discussion is important. In the revision, we will refocus Figure 7 on describing new results that we can learn. As outlined in the responses above, this paper reveals an important insight: the SDD model – with suitable modifications such as temporally restricted Bcd production – can explain all observed properties of Bcd gradient formation. Other mechanisms – such as *bcd *mRNA gradients – are not required.

    • The exposition of the manuscript would benefit significantly by including a section either in the introduction or the appropriate section of the results that defines the competing models for gradient formation. In the current version, these models are only cited, and the key details only come out late (e.g., lines 302 onward, in the Discussion). Nevertheless, some of the results are presented as if in dialog with these models, but it reads as a one-sided conversation. For instance: Figure 3. The undercurrent in this figure is the RNA-gradient model. In the context of this model, the results clearly show that translation of bcd is restricted to the anterior. Without this context, Figure 3 could read as a fairly unremarkable observation that translation occurs wherever there is mRNA. Restructuring the manuscript to explicitly name competing models and to address how experimental results support or detract from each competing model would greatly enhance the impact of the exposition.*

    We thank the reviewer for this suggestion. We will add the current models of Bcd gradient formation in the introduction section and will change the narrative of results in the section explaining the models.

    (4A) Related to point 3: The entire results text surrounding Figure 2 should be revised to include more detail about A) what specific hypotheses are being tested; and B) to critically evaluate the limitations of the experimental approaches used to evaluate these hypotheses. Hexanediol and high salt conditions are not named explicitly in the text, but the text touts these as "chemicals" that "disrupt P-body integrity." This implies that the treatments are specific to P-bodies. Neither of these approaches are only disrupting P Body integrity. This does not invalidate this approach, but the manuscript needs to state what hypothesis HD and NaCl treatment addresses, and acknowledge the caveats of the approach (such as the non-specificity and the assumptions about the mechanism of action for HD).

    We have made the following edits to resolve this point. Revised line 158: “To further show that bcd storage in P bodies is required for translational repression, we treated mature eggs with chemicals known to disrupt RNP granule integrity (31, 37, 69-72). Previous work has shown that the physical properties of P bodies in mature Drosophila oocytes can be shifted from an arrested to a more liquid-like state by addition of the aliphatic alcohol hexanediol (HD) (Sankaranarayanan et al., 2021, Ribbeck and Görlich, 2002; Kroschwald et al., 2017). While 1,6 HD has been widely used to probe the physical state of phase-separated condensates both in vivo and in vitro (Alberti et al., 2019; McSwiggen et al., 2019; Gao et al., 2022), in some cells it appears to have unwanted cellular consequences (Ulianov et al., 2021). These include a potentially lethal cellular consequences that may indirectly affect the ability of condensates to form (Kroschwald et al., 2017) and wider cellular implications thought to alter the activity of kinases (Düster et al., 2021). While we did not observe any noticeable cellular issues in mature Drosophila oocytes with 1,6 HD, we also used 2,5 HD, known to be less problematic in most tissues (Ulianov et al., 2021) and the monovalent salt sodium chloride (NaCl), which changes electrostatic interactions (Sankaranarayanan et al., 2021).”

    (4B) Continuing the comment above: it is good that the authors checked that HD and NaCl treatment does not cause egg activation. But no one outside of the field of Drosophila egg activation knows what the 2-minute bleach test is and shouldn't have to delve into the literature to understand this sentence. Please explain in one sentence that "if eggs are activated, then x happens following a short exposure to bleach (citations). We exposed HD and NaCl treated eggs to bleach and observed... ."

    We have made the following edits to resolve this point. Revised line 174: “After treating mature eggs with these solutions, we observed BcdSun32 protein in the oocyte anterior (Figure 2A-B). One caveat to this experiment could be that treating mature eggs with these chemicals results in egg activation which would in turn generate Bcd protein. To eliminate this possibility, we first screened for phenotypic egg activation markers, including swelling and a change in the chorion (73). We also applied the classic approach of bleaching eggs for two minutes which causes lysis of unactivated eggs (74). All chemically treated eggs failed this bleaching test meaning they were not activated (74). While we unable to rule out non-specific actions of these treatments, these experiments corroborate that storage in P bodies that adopt an arrested physical state is crucial to maintain *bcd *translational repression (31).”

    (4C) Continuing the comment above: The section of the results related to the endos mutation needs additional information. It is not apparent to the average reader how the endos mutation results in changes in RNP granules, nor what the expected outcome of such an effect would "further test the model" set up by the HD and NaCl experiments. The average reader needs more hand-holding throughout this entire section (related to figure 2) to follow the exposition of the results.

    We have made the following edits to resolve this point. Edited line 185: “Finally, we used a genetic manipulation to change the physical state of P bodies in mature oocytes. Mutations in Drosophila Endosulfine (Endos), which is part of the conserved phosphoprotein ⍺-endosulfine (ENSA) family (75), caused a liquid-like P body state after oocyte maturation, similar to that observed with chemical treatment (Figure 2C) (31). This temporal effect matched the known roles of Endos as the master regulator of oocyte maturation (75, 76). endos mutant oocytes lost the colocalisation of bcd mRNA and P bodies, concurrent with P bodies becoming less viscous during oocyte maturation (Figure 2D, Figure S1). Particle size and position analysis showed that bcd mRNA prematurely exhibits an embryo distribution in these mutants (Figure 2E). Due to genetic and antibody constraints, we are unable to test for translation of bcd in the endos mutant. However, it follows that bcd observed in this diffuse distribution outside of P bodies would be translationally active (Figure 2E-F).”

    • (4D) Continuing the comment above: The average reader also needs a better explanation of what hypothesis is being tested in Figure 1 with the pharmacological inhibition of calcium.

    We have made the following edits to resolve this point. Revised line 138: “We next sought to maintain the relationship between bcd mRNA and P bodies through egg activation. This would act as a control to further test if colocalisation of bcd to P bodies was necessary for its translational repression. Previous work has shown that a calcium wave is required at egg activation for further development (references to add Kaneuchi et al., 2015; York-Anderson et al., 2019; Hu and Wolfner, 2019). Chemical treatment with NS8593 disrupts this calcium wave, while other phenotypic markers of egg activation are still observed (58). Using NS8593 to disrupt the calcium wave in the activated egg, we show P bodies are retained during ex vivo egg activation (Figure 1E). In these treated eggs, bcd mRNA remains colocalised with the retained P bodies (Figure 1F). Based on these results and previous observations (31, 66), we hypothesised that the loss of colocalisation between bcd and P bodies correlates with bcd translation.”

    *It is unclear why Bcd translation could not be measured in the endos mutant background, but it would be necessary to measure Bcd translation in the endos background. If genotypically it is not possible/inconvenient to invoke the suntag reporter in the endos background, would it not be sufficient to immunostain against Bcd itself? Different Bcd antisera have recently been reported and distributed by the Wieschaus and the Zeitlinger groups.

    We have recently received the Bcd antibody from the Zeitlinger group. This has not been shown to work for immunostaining. It remains unclear if it will be successful in this capacity, but we are currently testing it and will include this experiment in the revision if successful.

    *Figure 4 overall is glorious, but there is a problem with panel C. What are the white lines? Why does the intensity for the green and magenta channel change abruptly in the middle of the embryo?

    These white lines divide the embryo into 4 compartments. We used this method to quantify the intensity of Bcd translation with respect to the bcd puncta. We will correct this image as there is a problem in formatting.

    *It is noted that neither the methods section or the supplement does not contain any mention of how the modeling was performed. How was parameter beta fit? At least a brief section should be added to the methods describing how beta was fit (pending adjustments suggested in comment 1 above). A platinum-level addition would include a modeling supplement that reports the sensitivity of model outcomes to changes in parameters.

    We apologise for this omission and will include full methodological details in the revision.

    Minor Comments:

      • Line 28: "Source-Diffusion-Degradation" should be changed to "Synthesis-..."* We will edit in the revised version.

    *Line 39: "blastocyst" should be "blastoderm stage embryo".

    We will edit in the revised version.

    • Line 81: "P bodies are an evolutionarily cytoplasmic RNP granule." is "conserved" missing here?

    We will edit in the revised version.

    • Throughout the manuscript, there should be better reporting of the imaged genotypes and whether the suntag is being visualized by indirect immunostaining of fixed tissues or through an encoded nanobody-GFP fusion.

    We will explain in detail in the revised version.

    • Figure 1G: Why is the background staining so different across conditions? Is this a normalization artifact?*__ __

    We agree with this shortcoming. We have now added the following to the figure legend to clarify this observation. “G: Representative fixed 10 µm Z-stack images (from 10 samples) showing BcdSun32 protein (anti-GCN4) is only present at the anterior of an in vitro activated egg or early embryo 30-minute post fertilization. BcdSun32 protein is not detected in these samples at the posterior pole (image contrast increased to highlight the lack of distinct particles at the posterior). BcdSun32 protein is also not detected at the anterior or posterior of a mature oocyte or an in vitro activated egg incubated with NS8953 (images have the contrast increased to highlight the lack of distinct particles). Scale bar: 20 mm; zoom 2 mm.” (Revised line 623).

    Figure 2 legend: what is +Sch in the x-axis labels of figure 2B? The legend says that 2B is the quantification of the data in 2A, but there is no (presumed control) +Sch image in 2A.__ __

    Thank you for this suggestion we have added the data to Figure 2A.

    • Figure 5A largely repeats information presented in figure 4A. Please consider moving to a supplement. Also, please re-orient embryos to follow the convention that dorsal-most surfaces be presented on the top of the displayed images.

    Thank you for this suggestion. We will consider moving Figure 5A to the supplementary.

    • The lower-case roman numerals referred to in the text for figure 7B are not included in the corresponding figure panel.

    We will edit in the revised version.

    • Figure 7C y-axis typo (concentration).

    We will edit in the revised version.

    • Line 222: "make a long-range functional gradient": more accurate to say, "but also marks mature, Bcd protein which resolves in the expected long-range gradient."

    We will edit in the revised version.

    • Methods: Please check that all buffers referred to as acronyms are both compositionally defined in the reagents table, and that full names are written out at the time of first mention in the presented order. For instance, Schneider's media is referred to a few times before defining the acronym about midway through the methods section.*__ __

    We have added to Figure 2B: “Quantification of experiments shown in A. The number of oocytes that displayed Bcd protein at the anterior as measured by the presence of BcdSun32 at the anterior of the oocyte, but not the posterior. Schneider’s Insect Medium (+Sch) used as a negative control. N = 30 oocytes for each treatment. Scale bar: 5 um.” (Revised line 646).

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    Referee #3

    Evidence, reproducibility and clarity

    This is a review of "Dynamics of bicoid mRNA localization and translation dictate morphogen gradient formation" by Athilingam et al. In this manuscript, the authors perform quantification of mRNA localization and translation of bicoid, spanning oogenesis through the maternal to zygotic transition, yielding a definitive characterization of Bicoid gradient formation. The experiments, analysis, and interpretation are on the whole performed rigorously. I very much enjoyed this paper, partly for incorporating the aspects of bcd regulation during oogenesis, which compared to embryonic function of bcd is relatively under-studied. Also valuable is improving the characterization of how bcd expression is shut down at NC14. I have several major comments for revision, and a few minor comments. I should stress that none of the major comments are terrible but are intended to improve the impact/readability/flow of this nice manuscript. With the exception of a straightforward immunostaining experiment, all major comments constitute reworking of the model or the text.

    Major Comments:

    1. It is not evident from the main results and methods text that the new SDD model incorporates the phenomenon reported in figure 4B. From my reading, the parameter beta accounts for the Bcd translation rate, which according to figure 7B(ii) effectively switches from off to on around fertilization and thereafter remains constant. Figure 4B shows that the fraction of bcd mRNA engaged in translation decreases beginning around NC12/13, and this is one of the more powerful results that comes from monitoring translation in addition to RNA localization/abundance/stability. My expectation based on figure 4B would be that parameter beta should decrease over time beginning around 90-100 minutes and approach zero by ~150 minutes. This rate could be fit to the experimental data that yields figure 4B. The modeling should be repeated while including this information.

    2. The presentation of the SDD model should be expanded to address how well the characteristic decay length fits A) measured Bcd protein distributions, B) measured at different nuclear cycles. This would strengthen the claim that the new SDD model better captures gradient dynamics given the addition of translation and RNA distribution. These experimental data already exist as reported in Figure 5. In the current Figure 7, panels D and D' add little to the story and could be moved to a supplement if the authors want to include it (in any case, please fix the typo on the time axis of fig 7D' to read "hours"). The model per cell cycle and the comparison of experimental and modeled decay lengths could replace current D and D'.

    3. The exposition of the manuscript would benefit significantly by including a section either in the introduction or the appropriate section of the results that defines the competing models for gradient formation. In the current version, these models are only cited, and the key details only come out late (e.g., lines 302 onward, in the Discussion). Nevertheless, some of the results are presented as if in dialog with these models, but it reads as a one-sided conversation. For instance: Figure 3. The undercurrent in this figure is the RNA-gradient model. In the context of this model, the results clearly show that translation of bcd is restricted to the anterior. Without this context, Figure 3 could read as a fairly unremarkable observation that translation occurs wherever there is mRNA. Restructuring the manuscript to explicitly name competing models and to address how experimental results support or detract from each competing model would greatly enhance the impact of the exposition.

    4A) Related to point 3: The entire results text surrounding Figure 2 should be revised to include more detail about A) what specific hypotheses are being tested; and B) to critically evaluate the limitations of the experimental approaches used to evaluate these hypotheses. Hexanediol and high salt conditions are not named explicitly in the text, but the text touts these as "chemicals" that "disrupt P-body integrity." This implies that the treatments are specific to P-bodies. Neither of these approaches are only disrupting P Body integrity. This does not invalidate this approach, but the manuscript needs to state what hypothesis HD and NaCl treatment addresses, and acknowledge the caveats of the approach (such as the non-specificity and the assumptions about the mechanism of action for HD).

    4B) Continuing the comment above: it is good that the authors checked that HD and NaCl treatment does not cause egg activation. But no one outside of the field of Drosophila egg activation knows what the 2-minute bleach test is and shouldn't have to delve into the literature to understand this sentence. Please explain in one sentence that "if eggs are activated, then x happens following a short exposure to bleach (citations). We exposed HD and NaCl treated eggs to bleach and observed... ."

    4C) Continuing the comment above: The section of the results related to the endos mutation needs additional information. It is not apparent to the average reader how the endos mutation results in changes in RNP granules, nor what the expected outcome of such an effect would "further test the model" set up by the HD and NaCl experiments. The average reader needs more hand-holding throughout this entire section (related to figure 2) to follow the exposition of the results.

    4D) Continuing the comment above: The average reader also needs a better explanation of what hypothesis is being tested in Figure 1 with the pharmacological inhibition of calcium.

    1. It is unclear why Bcd translation could not be measured in the endos mutant background, but it would be necessary to measure Bcd translation in the endos background. If genotypically it is not possible/inconvenient to invoke the suntag reporter in the endos background, would it not be sufficient to immunostain against Bcd itself? Different Bcd antisera have recently been reported and distributed by the Wieschaus and the Zeitlinger groups.

    2. Figure 4 overall is glorious, but there is a problem with panel C. What are the white lines? Why does the intensity for the green and magenta channel change abruptly in the middle of the embryo?

    3. It is noted that neither the methods section or the supplement does not contain any mention of how the modeling was performed. How was parameter beta fit? At least a brief section should be added to the methods describing how beta was fit (pending adjustments suggested in comment 1 above). A platinum-level addition would include a modeling supplement that reports the sensitivity of model outcomes to changes in parameters.

    Minor Comments:

    • Line 28: "Source-Diffusion-Degradation" should be changed to "Synthesis-..."
    • Line 39: "blastocyst" should be "blastoderm stage embryo".
    • Line 81: "P bodies are an evolutionarily cytoplasmic RNP granule." is "conserved" missing here?
    • Throughout the manuscript, there should be better reporting of the imaged genotypes and whether the suntag is being visualized by indirect immunostaining of fixed tissues or through an encoded nanobody-GFP fusion.
    • Figure 1G: Why is the background staining so different across conditions? Is this a normalization artifact?
    • Figure 2 legend: what is +Sch in the x-axis labels of figure 2B? The legend says that 2B is the quantification of the data in 2A, but there is no (presumed control) +Sch image in 2A.
    • Figure 5A largely repeats information presented in figure 4A. Please consider moving to a supplement. Also, please re-orient embryos to follow the convention that dorsal-most surfaces be presented on the top of the displayed images.
    • The lower-case roman numerals referred to in the text for figure 7B are not included in the corresponding figure panel.
    • Figure 7C y-axis typo (concentration).
    • Line 222: "make a long-range functional gradient": more accurate to say, "but also marks mature, Bcd protein which resolves in the expected long-range gradient."
    • Methods: Please check that all buffers referred to as acronyms are both compositionally defined in the reagents table, and that full names are written out at the time of first mention in the presented order. For instance, Schneider's media is referred to a few times before defining the acronym about midway through the methods section.

    Referees cross-commenting

    OK, We've been asked to comment on each others' reviews. I am reviewer 3. We have not been asked, as far as I can tell, to come up with a consensus review.

    Overall, I feel that we are all generally enthusiastic about this manuscript. From most to least enthusiastic, we have reviewer 1, 3, and finally 2. But all three of us are apparently advocating positively and encouraging revision and clarification because, as we all agree, these results are important to publish.

    Consensus Strengths:

    1. The experimental approach is elegant, rigorous, and innovative, especially the real-time visualization of Bcd translation.
    2. The data provide new mechanistic insight into when and where bcd is translated and how this changes over developmental time.
    3. The relocalization of bcd mRNAs to P bodies during nc14 and the implications for RNA degradation are particularly compelling.
    4. The manuscript establishes a path toward refining reaction-diffusion models of morphogen gradients using direct measurements of translation dynamics.

    I agree with all of Reviewer 1's minor points.

    I agree with Reviewer 2's points about:

    • Showing the SunTag validation data using the fluorescent reporter.
    • Clarifying the noted "translation" vs. "protein" issues. This bothered me too, but I wasn't able to articulate the issue as well as done here. This major issue summarizes several of the Reviewer's comments.
    • Generally tightening the precision with which the results are discussed.

    Overall: we have all provided favorable reviews that require mostly tightening of the text, showing some control datasets, maybe quantifying more points across the AP axis, and presenting the SDD model more comprehensively (comparing with old/translation-agnostic model, reporting characteristic decay lengths at different nuclear cycles, incorporating the reported change in translation rate across nuclear cycles (if this survives the clarification of what 'translation' means per Reviewer 2's comments), and perhaps providing more methodological detail on how parameters were fit).

    Significance

    The importance of this study is at several levels. For the developmental biologist, it addresses important mechanisms of translational control and RNA stability over the functional lifetime of a single, critical biological cue that governs embryonic patterning. Not only do the experiments provide quantification of these features, but also point to likely candidates (P-bodies) for gating bcd's translation in the narrow window between egg activation and cellular blastoderm. For the biophysically-inclined, this adds critical quantitative information of translational state that allows for further refining computational models for how this manifestation of a reaction-diffusion system actually comes together in a complex biological context.

    The primary audience for this work will be the two groups above: developmental biologists and scientists interested in the quantitative modeling of biological phenomena.

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    Referee #2

    Evidence, reproducibility and clarity

    In this manuscript by Athilingam et al., the authors are studying the translation of the morphogen Bicoid (Bcd), which is in anterior-posterior patterning of the blastoderm Drosophila embryo. They have used an array of sunTag elements in the 5' UTR of Bcd to detect the localization of translation. They found that, not only is Bcd not translated until egg activation, but it can only be translated at the anterior pole, even though bcd mRNA has a broader spatial distribution.

    In general, the paper uses a cutting-edge methodology to address one of the foundational questions of the best-studied morphogen gradient: namely, what is the spatial distribution of the Bcd source? Together with the dynamics of its spreading (which they addressed in a separate study in 2024) and Bcd degradation, their results point to a modified form of the synthesis/diffusion/degradation (SDD) model of Bcd gradient formation, which they have analyzed in the final subsection of the results. However, there are several major issues that erode the validity and impact of the paper, most of which can be put into the category of vague explanations, missing information, or contradictory statements, making it hard to understand/verify what conclusions can be drawn. This is also coupled with vague figures and captions. We describe these, and a few minor issues, in detail below:

    • Line 114: The authors claim to have validated the SunTag using a fluorescent reporter, but do not show any data. Ref 60 is a general reference to the SunTag, and not the Bcd results in this paper. Perhaps place their data into a supplemental figure or movie?
    • Line 128 and Fig. 1E: The claim that bcd becomes dispersed is difficult to verify by looking at the image. The language could also be more precise. What does it mean to lose tight association? Perhaps the authors could quantify the distribution, and summarize it by a length scale parameter? This is one of the main claims of the paper (cf. Line 23 of the abstract) but it is described vaguely and tersely here.
    • Line 146, Fig. 1G: This is a really important figure in the paper, but it is confusing because it seems the authors use the word "translation," when they mean "presence of Bcd protein." In other places in the paper, the authors give the impression that "bcd translation" means translation in progress (assayed by the colocalization of GCN4 and bcd mRNA). However, in Fig. 1G, the focus is only on GCN4. Detecting Bcd protein only at the anterior does not mean that translation happens only at the anterior (e.g., diffusion or spatially-restricted degradation could be in play).

    It would also be helpful to show a plot with quantification of Bcd detection (or translation) on the y-axis and a continuous AP coordinate on the x-axis, instead of just two points (anterior and posterior poles, the latter of which is uninteresting because observing no Bcd at the posterior pole is expected).

    Another issue with Fig. 1G is that the A and P panels presumably have different brightness and contrast. If not, just from looking at the A and P panels, the conclusion would be that Bcd protein is diffuse (and abundant) in the posterior and concentrated into puncta in the anterior. The authors should either make the brightness and contrast consistent or state that the P panel had a much higher brightness than the A panel.

    • Line 176: This section is very confusing, because at this point the authors already addressed the spatial localization of translation in Fig. 1G,H (see my above comment). However, here it seems the authors have switched the definition of translation back to "translation in progress." Therefore, the confusion here could be eliminated by addressing the above point.
    • Line 185: The sentence here is seemingly contradictory: "most...within 100 microns" implies that at least some are beyond 100 microns, while the sentence ends with "[none]...more than 100 microns." The language could perhaps be altered to be less vague/contradictory.
    • Line 204: It would be really nice to have quantification of the translation events, such as curves of rate of translation as a function of a continuous AP coordinate, and a curve for each nc.
    • Line 209 and Fig 4C: The authors use the terms "intensity of translation events" or "translation intensity" without clearly defining them. From the figure (specifically from the y-axis label), it looks like the authors are quantifying the intensity per molecule (which is not clearly the same thing as "translation intensity"), but it would be nice if that were stated explicitly.

    In addition, the authors again quantify only two points. This is a continuously frustrating part of the manuscript, which applies to nearly all figures where the authors looked only at two points in space. At a typical sample size of N = 3, it seems well within time constraints to image at multiple points along the AP axis.

    Furthermore, it sounds like the authors are saying the "translation intensity" is the same in anterior and the posterior, which is counterintuitive. The expectation is that translation would be undetectable at the posterior end, in part because bcd mRNA would not be present. (Note that this expectation is even acknowledged by the authors on Line 185, which I comment on above, and also on Line 197). There should also be very low levels of Bcd protein (possibly undetectable) at the posterior pole. As such, the authors should explain how they think their claim of the same "translation intensities" in the anterior vs posterior fits into the bigger picture of what we know about Bcd and what they have already stated in the manuscript. They should also explain how they observed enough molecules to quantify at the posterior end. The authors should also disclose how many points are in each box in the boxplot. For example, the sample size is N = 3 embryos. In just three embryos, how many bcd/GCN4 colocalizations did the authors observe at the posterior end of the embryo?

    • Line 215: The sentence that starts on this line seems self-contradictory: I cannot tell whether or not there is a difference in translation based on position.
    • Line 229: Long-ranged is a relative term. From the graph, one could state there is some spatial extent to the mRNA gradient, so it is unclear what the authors mean when they say it is not "long-ranged." Could the mRNA gradient be quantified, such as with a spatial length scale? This would provide more information for readers to make their own conclusions about whether it is long-ranged.
    • Line 230: When the authors claim the Bcd gradient is steeper earlier, a quantification of the spatial extent (exponential decay length scale) would be appropriate. Indeed, lambda as a function of time would be beneficial. It should also be placed in context of earlier papers that claim the spatial length scale is constant.
    • Lines 235-236: The two sentences that start on these two lines are vague and seemingly contradictory. The first sentence says there is a spatial shift, but the second sentence sounds like it is saying there is no spatial change. The language could be more precise to explain the conclusions.

    Minor issues/typos (still must be addressed for content):

    • Line 81: Probably meant "evolutionarily conserved"
    • Figure 1 legend: part B says "from 15 samples" but also says N = 20. Which is it, or do these numbers refer to different things?
    • Line 217: migration of what?
    • Line 228: "early embryo" is vague. The authors should give specific time points or nuclear cycle numbers.
    • Line 301: Other locations in the paper say 75 microns or 100 microns.
    • Fig. 5: all images should be oriented such that the dorsal midline is on the upper half of the embryo/image.
    • Fig. 5B: There are light tan and/or light orange curves (behind the bold curves) that are not explained.
    • Fig. 5C: the plot says "normalized" but nowhere do the authors describe what the curves are normalized to. There is also no explanation for what the broad areas of light color correspond to.

    Referees cross-commenting

    This is Reviewer 2. Yes, I am enthusiastic about the work: it is a much needed set of experiments and it fits well into the overall goal of quantitatively understanding the processes that establish the Bcd gradient. My main concern(s) about this paper is the loose and vague way they described their experiments and the interpretations. My hope is they will use the revision as an opportunity to more precisely explain their work.

    Other than that, I am in agreement with the other reviewers on the need to revise for clarity and publish this important work.

    Significance

    The results, if upheld, are highly significant, as they are foundational measurements addressing a longstanding question of how morphogen gradients are formed, using Bcd (the foundational morphogen gradient) as a model. They also address fundamental questions in genetics and molecular biology: namely, control of mRNA distribution and translation.

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    Referee #1

    Evidence, reproducibility and clarity

    In this paper the authors use the Suntag system to visualise bcd mRNA translation in the Drosophila embryo. They elucidate the relationship between bcd mRNA translation and P body localisation. In the oocyte, bcd mRNAs are localised in P bodies and translationally repressed, but upon egg activation bcd mRNAs are released from P bodies and translated. In addition, during mid-nc14, bcd mRNAs become localised to embryonic P bodies and degraded. The authors use their data to modify the Synthesis, Diffusion, Degradation model of Bcd gradient formation, which recapitulates the Bcd gradient detected experimentally.

    Overall, I think the data are of high quality and support the authors' conclusions. I only have minor comments, as follows:

    Fig 1B - add arrows showing mRNAs being translated or not (the latter mentioned in line 113 is not so easy to see).

    Fig 2A - add a sentence explaining why 1,6HD, 2,5HD and NaCl disrupt P bodies.

    Fig 4C - explain in the legend what the white lines drawn over the image represent. And why is there such an obvious distinction in the staining where suddenly the DAPI is much more evident (is the image from tile scans)?

    Line 215 - 'We did not see any significant differences in the translation of bcd based on their position, however, there appears an enhanced translation of bcd localised basally to the nuclei (Figure S5).' Since the difference is not significant, I do not think the authors should conclude that translation is enhanced basally.

    Line 218: 'The interphase nuclei and their subsequent mitotic divisions appeared to displace bcd towards the apical surface (Figure S6B).' Greater explanation is needed in the legend to Fig S6B to support this statement as the data just seem to show a nuclear division - I would have thought an apical-basal view is needed to conclude this.

    Fig 5B - the authors compare Bcd protein distribution across developmental time. However, in the early time points cytoplasmic Bcd is measured (presumably as it does not appear nuclear until nc8 onwards) and compare the distribution to nuclear Bcd intensities from nc9 onwards. Is most/all of the Bcd protein nuclear localised form nc9 to validate the nuclear quantitation? Does the distribution look the same if total Bcd protein is measured per volume rather than just the nuclear signal? Are the authors assuming a constant fast rate of nuclear import?

    Line 259 - 'We then asked if considering the spatiotemporal pattern of bcd translation' - the authors should clarify what new information was included in the model. Similarly in line 286, 'By including more realistic bcd mRNA translation' - what does this actually mean? In line 346, 'We see that the original SDD model .... was too simple.' It would be nice to compare the outputs from the original vs modified SDD models to support the statement that the original model was too simple.

    Fig S1A - clarify what the difference is between the 2 +HD panels shown.

    Fig S2E - the graph axis label/legend says it is intensity/molecule. Since intensity/molecule is higher in the anterior for bcd RNAs, is this because there are clumps of mRNAs (in which case it's actually intensity/puncta)?

    Fig S4 - I think this line is included in error: '(B) The line plots of bcd spread on the Dorsal vs. Ventral surfaces.' In B, D, E - is the plot depth from the dorsal surface? I would have preferred to see actual mRNA numbers rather than normalised mRNAs. In Fig S4D moderate, from 10um onwards there are virtually no mRNA counts based on the normalised value, but what is the actual number? The equivalent % translated data in Fig S4E look noisy so I wonder if this is due to there being a tiny mRNA number. The same is true for Figs S4D, E 10um+ in the low region.

    Referees cross-commenting

    I think the concerns raised by reviewers 2 and 3 are valid, and that it is feasible for the authors to address all the reviewers' concerns in order to improve the manuscript.

    Significance

    General assessment

    Strengths are: 1) the data are of high quality; 2) the study advances the field by directly visualising Bcd mRNA translation during early Drosophila development; 3) the data showing re-localisation of bcd mRNAs to P bodies nc14 provides new mechanistic insight into its degradation; 4) a new SDD model for Bcd gradient formation is presented. Limitations of the study are: 1) there was already strong evidence (but no direct demonstration) that bcd mRNA translation was associated with release from P bodies at egg activation; 2) it is not totally clear to me how exactly the modified SDD model varies from the original one both in terms of parameters included and model output.

    Advance

    The advance is conceptual, technical and mechanistic.

    Audience

    The results will be important to a broad range of researchers interested in the formation of developmental morphogen gradients and the post-transcriptional regulation of gene expression, particularly the relationship with P bodies.

    My expertise

    Wetlab developmental biologist

  5. Version published to 10.1101/2024.11.11.622966 on bioRxiv