miR-184 modulates dilp8 to control developmental timing during normal growth conditions and in response to developmental perturbations

  1. Jervis Fernandes
  2. Muhammed Naseem
  3. Ayisha Marwa MP
  4. Jishy Varghese

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Abstract

Organismal development depends on the precise coordination of growth and developmental timing, which is regulated by a complex interplay of intrinsic and extrinsic factors. However, the mechanisms underlying this regulation are not fully understood. Post-transcriptional regulation by microRNAs (miRNAs) plays a pivotal role in ensuring the proper timing of gene expression necessary for growth and development. In this study, we conducted a genetic screen to identify microRNAs that regulate developmental timing in Drosophila . Our screen identified miR-184, previously implicated in germline maturation and embryonic development, as a regulator of pupariation timing by acting in the larval imaginal discs. Using genetic and molecular approaches, we identified Drosophila insulin-like peptide 8 ( Dilp8 ), a paracrine factor critical for regulating developmental stability, as a target of miR-184 . During normal larval development miR-184 facilitates timely pupariation by regulating dilp8 levels. Furthermore, we demonstrate that miR-184 plays a critical role in tissue damage responses by inducing dilp8 expression, which delays pupariation to enable damage repair mechanisms. These findings reveal a novel post-transcriptional regulatory mechanism that links miR-184 to the control of developmental timing under normal growth conditions and in response to tissue damage.

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    Reviewer #1

    Evidence, reproducibility and clarity

    SUMMARY

    In this study, Fernandes and colleagues addressed the question of the role of micro-RNAs in regulating the coupling between organ growth and developmental timing. Using Drosophila, they identified the conserved micro-RNA miR-184 as a regulator of the developmental transition between juvenile larval stages and metamorphosis. This transition is under the control of the steroid hormone Ecdysone, and has been shown to be modulated in case of abnormal tissue growth to adjust the duration of larval growth in response to developmental perturbations. The relaxin-like hormone Dilp8 has been identified as a key secreted factor involved in this coupling. Here, the authors show that miR-184 is involved in the regulation of Dilp8 expression both in physiological conditions and upon growth perturbation. They propose that this function is carried out in imaginal tissues, where miR-184 levels are modulated by tissue stress. While several factors have already been involved in triggering sharp dilp8 induction at the transcriptional level, this study adds another level of complexity to the regulation of Dilp8 by proposing that its expression is fine-tunned post-transcriptionally through repression by miR-184.

    __MAJOR COMMENTS______

    Overall, the manuscript is well organized, and the logics of the experimental plan well presented. The results are clear, and I appreciate the quality of the pupariation curves. However, I believe that two main conclusions of the paper are not fully supported by the results presented in the figures: the direct regulation of dilp8 3'UTR by miR-184, and the specificity of this regulation in imaginal discs. Here I develop in more details these two aspects.

    Comment 1) The strategy of the 3'UTR sensor is not fully optimized. Indeed, in most experiments, qRT-PCR is used to assess dilp8 expression levels, although it reflects both transcriptional and post-transcriptional. Importantly, to show that post-transcriptional regulation is involved in the response to tissue damage, the levels of the 3'UTR sensor should be analyzed in discs expressing RAcs (showing at the same time that the response is cell-autonomous in the discs). The expected upregulation of the sensor should be prevented by simultaneous expression of miR-184. This approach would shed light on the relative contribution of transcriptional versus post-transcriptional regulation of dilp8 in response to growth perturbation.

    Response: We thank the reviewer for this comment. We agree that qRT-PCRs do not distinguish between transcriptional and post-transcriptional changes of dilp8 levels, in response to changes in miR-184 levels and tissue damage. In addition to the qRT-PCR data we have looked at dilp8-3’UTR-GFP reporter in response to overexpression of miR-184 in the wingdisc using patched-Gal4 driver, which show downregulation of the GFP reporter in the ptc domain (Fig 4C-D’). This suggests that dilp8 mRNA is a direct target of miR-184 by post-transcriptional regulation through its 3’UTR. Further, to confirm the specificity of the effect of miR-184 on dilp8-3’UTR, we generated a dilp8-3’UTR mutant in which the single target site for miR-184 was mutated. We show that the mutated dilp8-3’UTR reporter doesn’t show any regulation in response to miR-184 overexpression in the ptc domain of the wingdisc (Fig. 4E, E’, F, F’). This experiment confirms the specificity of the dilp8-3’UTR regulation by miR-184.

    As suggested by the reviewer we analysed dilp8-3’UTR-GFP reporter expression by overexpressing RicinA using ptcGAL4 driver in the wing imaginal disc (Fig. S6F-G’). We observed a slight but consistent increase in the dilp8-3’UTR-GFP reporter expression, indicating post-transcriptional regulation of dilp8 expression in response to tissue damage. However, the increase of reporter GFP levels observed in this experiment in response to tissue damage is mild (Fig. S6F-G’) than expected based on the qRT-PCR results (Fig S6A and B). We have added this new data to the manuscript (Fig. S6F-G’).

    We propose the following reasons to explain this result:

    a) both transcriptional and post-transcriptional regulation of dilp8 mRNA in response to developmental perturbations

    b) the data on 3’UTR reporter GFP is specifically from the ptc domain expression of RicinA, whereas for dilp8 transcript levels we have expressed RicinA in all larval imaginal tissues, or in the entire wing imaginal disc, which could be one of the reasons for the stronger effect seen on dilp8 mRNA levels

    c) we are not certain if the tubulin-promoter driven dilp8-3’UTR GFP reporter reflects post-transcriptional regulation of dilp8 by miR-184 efficiently in comparison to qRT-PCR. This is especially as the reporter-GFP-3’UTR will be expressed at very high levels due to the tubulin promoter, a majority of this reporter-GFP mRNA may not be relieved from degradation due to the moderate suppression of miR-184 in response to RicinA overexpression.

    Thus, our experiments suggest that dilp8 levels are regulated post-transcriptionally by miR-184 which contributes to pupariation delays in response to tissue damage. In support of this, we could rescue pupariation delays and dilp8 induction caused by RicinA expression using overexpression of *miR-184 *(Figs 5B, C). Thus, we confirm that the effect of post-transcriptional regulation by miR-184 during developmental perturbations also contributes to dilp8 induction and pupariation delays. Unfortunately, due to experimental limitations we could not perform simultaneous expression of RicinA and miR-184 to evaluate the rescue of dilp8-3’UTR-GFP sensor expression. The levels of dilp8-3’UTR sensor GFP is reduced efficiently by miR-184 overexpression (Fig 4D), which prevented us from attempting the rescue of the moderate increase of dilp8-3’UTR GFP levels in response to RicinA.

    Comment 2) In my opinion, the use of a 3'UTR sensor is not sufficient to conclude that the regulation by miR-184 is direct, as miR-184 could also regulate an intermediate factor that acts on dilp8 post-transcriptional regulation. To solve this issue, a common strategy is to generate a 3'UTR sensor with mutated binding sites that should abolish the regulation by miR-184. This mutated 3'UTR might also respond differently to tissue damage, which would strongly support the conclusions of the study.

    Response: We couldn’t agree more with the reviewer, this comment is addressed in the response to comment 1. We have confirmed the specificity of regulation of dilp8-3’UTR by miR-184 using target site mutated dilp8-3’UTR (new figures added to the manuscript Fig. 4E, E’, F, F’). We tested if the changes in dilp8 mRNA levels in response to tissue damage is post-transcriptional mediated by miR-184. We observe that there is a slight, but consistent increase of dilp8-3’UTR GFP reporter levels in the ptc domain of wingdisc in response to RicinA expression, suggesting a role for miR-184 mediated post-translational regulation of dilp8. However, we have not yet tested the mutated dilp8-3’UTR GFP reporter in response to tissue damage.

    Comment 3) Concerning the tissue-specific regulation of Dilp8 by miR-184, these results need to be strengthened. Indeed, this comes mostly from phenotypes observed with rn-GAL4. Although this is a classical tool for driving expression in imaginal discs, rn-GAL4 also drives strong expression in other tissues that could contribute to triggering a delay, such as the CNS and part of the gut (proventriculus). In our hands, some growth phenotypes in the wing obtained with rn-GAL4 could be fully reverted by blocking GAL4 in the CNS indicating that the phenotype was not wing-specific. Importantly, miR-184 seems to be highly expressed in the CNS according to FlyBase, reinforcing the possibility that it plays a role in this organ. Here I propose approaches to confirm that miR-184 mediated regulation of dilp8 and developmental timing indeed occur in the discs:

    - Another driver with less secondary expression sites could be used (pdmR11F02-GAL4), or rn-GAL4 could be combined with an elav-GAL80 to prevent expression in most neurons. - The authors could identify the source of Dilp8 upregulation in miR-184 mutants using tissue-specific qRT-PCR instead of whole larvae expression like in Fig 4A-B. - This tissue-specific upregulation could be functionally tested using a rescue experiment, in which the delay observed in miR-184 mutants could be rescued by disc-specific downregulation of Dilp8 (using pdm2-GAL4 for instance).

    Response: We are thankful to the reviewer, and agree that it is important to show that the effects that we see using rn-Gal4 are specific to imaginal discs, and not due to an effect in CNS. We tested this by expressing miR-184 sponge in the CNS. Though miR-184 is highly expressed in the larval CNS, downregulation of miR-184 specifically in the pan-neuronal background using elav-GAL4 led to no effects on pupariation timepoint. We have added this as supplementary data Figure S4. Therefore, we believe that the miR-184 downregulation phenotype in the rnGAL4 background can be mainly attributed to its role in the imaginal discs. In addition, as suggested by the reviewer we have also demonstrated that downregulation of miR-184 in the imaginal discs using rnGAL4 driver leads to an increase in dilp8 expression (Fig S5B). Thus confirming that dilp8 mRNA is enhanced in the imaginal discs by blocking miR-184.

    OPTIONAL: Because it is known that dilp8 is strongly regulated at the transcriptional level, the relative input from post-transcriptional upregulation is an important question arising from this study. Although it might be a more long-term approach, I believe that generating a Dilp8 mutant lacking its 3'UTR or, even better, with mutated miR-184 binding sites, would shed light on the role of this regulation for the response to growth perturbation and/or developmental stability (fluctuating asymmetry).

    Response: We thank the reviewer for the suggestion. This would have been an interesting experiment to carry out especially in the context of fluctuating asymmetry.

    MINOR COMMENTS

    __ I think that a number of results could be moved to SI as they are either controls, or reproduce published data without bringing novelty. For instance, results in Fig 5A-D are similar to data published by Sanchez et al, as stated in the text. Fig6A as well.__

    __Response: __We thank the reviewer for this suggestion, Fig. 5A-D, and F has been moved to Fig. S6A-E. We have also moved data from Fig. 6 to Fig. 5, as a result Fig 6 A-D has become Fig. 5 B-D.

    __ Fig 6D is quite mysterious, as it suggests that basal JNK activation regulates miR-184, which is different from a context of tissue damage. I think that this result could be removed. Alternatively, if the authors want to dig in that direction, more experiments should be provided, such as bskDN expression in an RAcs context and the effects on miR-184 levels and the 3'UTR sensor (since transcript levels are already published).__

    Response: We would like to clarify that our experiments suggest that endogenous JNK signalling negatively regulates miR-184, as blocking basal JNK signalling using bskDN increased the levels of miR-184 (changed to Fig 5D). Enhanced JNK signalling has been reported to be involved in tissue damage responses, and we propose that RicinA mediated increase in JNK signalling leads to the reduction of miR-184 (changed to Fig 5A, S6D-E). However, we are not strongly implying this as we did not co-express RicinA and bskDN to show that JNK signalling is responsible for the drop in miR-184 levels in response to tissue damage. We thank the reviewer for seeking this explanation, we have rewritten the results section to improve clarity.

    __ The references related to Dilp8 should be checked more in detail in the intro and discussion. About Dilp8 and developmental stability: remove the ref to Colombani et al 2012, instead put Boone et al 2016 and add Blanco-Obregon et al 2022 (in addition to Garelli et al 2012 who initially identified this phenotype. About Lgr3 as the receptor for Dilp8: add Colombani et al, Current Biology 2015, and cite here Vallejo et al 2015, Garelli et al 2015. Among the important transcriptional regulators of Dilp8, Xrp1 could be mentioned (Boulan et al 2019, Destefanis et al 2022) as it plays a complementary function to JNK depending on the type of tissue stress.__

    __Response: __We are really sorry for the glaring errors in citing appropriate references. We thank the reviewer for correcting this for us. We have made necessary changes to the text.

    Significance

    GENERAL ASSESSMENT This study provides convincing data showing that the conserved microRNA miR-184 plays a role in regulating developmental timing in Drosophila through modulating the levels of Dilp8, a key factor in the coupling between tissue growth and developmental transitions. The results are convincing, but the general conclusions of the paper need to be strengthened regarding the direct regulation of dilp8 by miR-184 and the tissue-specificity of this interaction.

    ADVANCE Dilp8 is a key factor that modulates growth and timing in response to developmental perturbations and contributes to developmental precision in physiological conditions. As such, its regulation has been studied by different groups in the last decade, leading to the identification of several inputs for its transcriptional regulation. Here, the authors uncover a post-transcriptional regulation by miR-184, adding another level of regulation of Dilp8 that contribute to ensuring proper regulation of developmental timing, and opening the possibility that miR-184 might play similar roles in other species.

    AUDIENCE This study is of interest for researchers in the field of basic science, with a focus on developmental timing, tissue damage and biological function of microRNAs.

    REVIEWER EXPERTISE Drosophila, growth control, developmental timing, Dilp8.

    Reviewer #2

    Evidence, reproducibility and clarity

    Drosophila has helped to characterize the mechanisms that coordinate tissue growth with developmental timing. The insulin/relaxin-like peptide Dilp8 has been identified as a key factor that communicates the abnormal growth status of larval imaginal discs to neuroendocrine neurons responsible for regulating the timing of metamorphosis. Dilp8, derived from imaginal discs, targets four Lgr3-positive neurons in the central nervous system, activating cyclic-AMP signaling in an Lgr3-dependent manner. This signaling pathway reduces the production of the molting hormone, ecdysone, delaying the onset of metamorphosis. Simultaneously, the growth rates of healthy imaginal tissues slow down, enabling the development of proportionate individuals.

    In this manuscript "miR-184 modulates dilp8 to control developmental timing during normal growth conditions and in response to developmental perturbations" by Dr. Varghese and colleagues, the authors identify a new post transcriptional regulator of Dilp8. The authors show that miR-184 plays a pivotal role in tissue damage responses by inducing dilp8 expression, which in turn delays pupariation to allow sufficient time for damage repair mechanisms to take effect.

    Major points:

    Comment 1) In most of the experiments for percentage of pupariation, the 50% pupariation in control is around 110 hours AED in figures 1, 2 and 3. In figures 5 and 6 using the UAS Ricin, the controls are more around 90 hours AED. Why this discrepancy?

    Response: We thank the reviewer for asking for this clarification. The former experiments for Figs 1-3 were carried out at 25oC while the latter experiments with a cold sensitive version of RicinA (UAS-RAcs), Figs 5 and 6 (now changed to Figs. 5 and S6 as suggested by reviewer #1) were carried out at 29oC (permissive temperature). This difference in temperature has led to alterations in pupariation timing. We apologise for not having mentioned this in the text, now we have made necessary corrections to the methods section clearly indicating this.

    Comment 2) What is the mechanism behind the expression of miR-184 in stress conditions? Is miR-184 also implicated in other conditions giving rise to a developmental delay (X-rays irradiation or animal bearing rasV12, scrib-/- tumors)?

    Response: We thank the reviewer for these questions.

    a) In response to developmental perturbations by RicinA, we believe that activation of JNK signalling controls miR-184 expression. We propose this as our experiments show that imaginal disc damage leads to enhancement of JNK signalling and increase in dilp8 mRNA levels (as reported earlier by Colombani et al 2012; Sánchez et al 2019), and a simultaneous reduction of miR-184 (Figs. S6A, D, E). We also have performed new experiments to show that in response to RicinA expression in the wingdisc there is moderate increase in the dilp8-3’UTR-GFP sensor expression (Figs. S6F-G’), indicating a post-transcriptional regulation of dilp8 expression in response to tissue stress. We also show that RicinA induced dilp8 expression and pupariation delay can be rescued by increasing miR-184 levels (Fig 5B and C), suggesting that the reduction of miR-184 in response to tissue damage contributes to the damage responses. In a separate experiment we show that blocking the endogenous JNK pathway by the expression of bskDN enhances miR-184 levels, suggesting that miR-184 is under the regulation of JNK signalling (Fig 5D). Hence, we speculate that during tissue stress, activation of JNK signalling leads to a reduction of miR-184 levels which contributes to regulating the levels of dilp8 post-transcriptionally and resulting in pupariation delays. The text has been modified to explain this better.

    b) In a previous paper by Shu et al., 2017 (https://doi.org/10.18632/oncotarget.22226) decreased expression of miR-184 was observed in a lglRNAi; RasV12 tumor background. Apart from this various studies have shown that dilp8 levels increase in response to tumour, radiation stress, apoptosis, and tissue damage (Yeom et al 2021, Ray et al 2019, Demay et al 2014, Katsuyama et al 2015, Colombani et al 2012, Garelli et al 2012). Whether the regulation of dilp8 by miR-184, occurs in these backgrounds is yet to be tested. We have now discussed this possibility in the manuscript.

    Comment 3) dilp8 mutant animals have also been shown to be more resistant to starvation or desiccation (https://doi.org/10.3389/fendo.2020.00461). Is miR-184 implicated in this answer?

    Response: We thank the reviewer for this question. In our earlier experiments miR-184 has been demonstrated to be regulated by nutrition in the larval stages and lack of miR-184 led to enhanced larval death in response to diet restriction (Fernandes et al., 2022). miR-184 was also demonstrated to play a role in the insulin producing cells (IPCs) in regulating lifespan (Fernandes & Varghese., 2022). In the current work, we propose miR-184 to act upstream of dilp8 in response to stress stimuli. Hence, it is possible that miR-184 might be involved in responses to starvation and desiccation stress in the adult female flies, by regulating dilp8 levels post-transcriptionally. However, it has not been tested yet if the miR-184 regulation of dilp8 plays a role in resistance to starvation or desiccation in adult females, as this was not within the scope of the current study. We have now added this reference in the discussion section.

    Comment 4) dilp8 expression has been also shown to be regulated by Xrp1 in response to ribosome stress (https://doi.org/10.1016/j.devcel.2019.03.016). This paper should be included in the manuscript. Is it possible that the expression levels of miR184 are regulated by Xrp1?

    Response: We thank the reviewer for the suggestion and have incorporated the reference into the paper. During ribosome stress in the larval imaginal discs the stress-response transcription factor Xrp1 acts through dilp8 in regulating systemic growth. We agree with the reviewer, it is possible that expression of miR-184 is regulated by Xrp1. Currently we have not explored this possibility. We have now added this to the discussion section.

    Minor points:

    __ Does the overexpression of miR184 induce an increased fluctuating asymmetry?__

    Response: We thank the reviewer for asking this question. The role of dilp8 in the fluctuation asymmetry is only observed in the dilp8 hypomorphic mutant background. To replicate this we would have to overexpress miR-184 in either the whole larvae or in the wing discs. Unfortunately overexpression of miR-184 in the wing discs (using rnGAL4) leads to pupal lethality while as overexpression of miR-184 in the whole larvae leads to embryonic lethality and therefore we were not be able to conclude from our experiments if miR-184 overexpression induces increased fluctuating asymmetry.

    2. There are 2 references Colombani et al. (2012 for Dilp8 and 2015 for Lgr3). Can you double check that they are used accordingly

    Response: We thank the reviewer for pointing these errors out and we have incorporated these changes into the paper.

    Significance

    Altogether, the paper present compiling lines of evidence supporting the proposed model. The experiments are well designed and are convincing. The papers is interesting and relevant for a broad audience.

    __Reviewer #3 __

    Evidence, reproducibility and clarity (Required):

    This is an interesting study demonstrating an interaction between miR-184 and the Drosophila insulin-like peptide 8 (dilp8) in the tissue damage response. The authors show that Dilp8 activity is negatively regulated by miR-184, apparently through direct interaction between miR-184 and the dilp8-3'UTR, which leads to lower dilp8 mRNA transcript levels, via an undetermined mechanism, supposedly its degradation? Furthermore, the authors show that during aberrant tissue growth, miR-184 levels are very slightly downregulated (see comment below), and based on other experiments, imply causation of this with the increased dilp8 mRNA levels that occur in these tissues, again via an unclear mechanism: upregulation or stabilization of dilp8 mRNA. The authors present evidence that the JNK pathway, which had been known to be critical for dilp8 mRNA upregulation upon tissue damage, does so via miR-184.

    Major Comments:

    __Comment 1: The data showing the direct regulation of dilp8-3'UTR by miR-184 are not very strong and would require more controls to strengthen the claim, as described below. __

    Response: We have performed new experiments to validate that dilp8-3’UTR is regulated by miR-184. Please see the detailed responses to comments 10-12 below.

    __Comment 2: The miR-184 effects are also very small (less than 2-fold reduction with tissue damage; or less than 2-fold induction with JNK-pathway inhibition via bskDN). These two points are the weakest part of the manuscript and model. __

    Response: We agree with the reviewers on this point. The reduction in miR-184 levels in response to RicinA expression is modest (25–30%), and the induction of miR-184 in response to bskDN expression is less than two-fold (Figs. 5A and D). In contrast, dilp8 transcript levels increase several-fold in response to RicinA expression (Fig. 5C, S6A and B). Since we measure dilp8 transcript levels by qPCR, we detect both transcriptional and post-transcriptional contributions to dilp8 regulation. In addition, we have performed a new experiment to check the post-transcriptional regulation of dilp8, in response to tissue damage. Though the change in the dilp8-3′UTR GFP reporter upon RicinA expression in the ptc domain of the wingdisc is mild (Figs. S6F-G’), this strongly suggests a post-transcriptional outcome of the reduction of miR-184 levels on dilp8. Hence, we propose that tissue damage induces strong transcriptional activation of dilp8, while the reduction of miR-184, despite its smaller magnitude, contributes to dilp8 upregulation via post-transcriptional regulation. In support of this, our experiments demonstrate direct regulation of the dilp8-3′UTR by miR-184 (Figs. 4C-F’), and show strong dilp8 mRNA upregulation in miR-184 deficient conditions (Fig. 4A and B), suggesting the role of miR-184 in maintaining dilp8 levels. We also show that RicinA induced effects on dilp8 and pupariation delay are reversed by co-expression of miR-184 (Fig. 5C). We do not claim that regulation by miR-184 is the sole mechanism for driving dilp8 induction during tissue damage, but suggest that miR-184-mediated post-transcriptional regulation acts in a complementary manner to transcriptional responses. Furthermore, we believe that the mild effect of JNK signaling on miR-184 (as shown by the bskDN experiment) is sufficient for the moderate reduction of miR-184 in response to tissue damage.

    Comment 3: ____Regarding the expression levels, it does not help that the authors show bar graphs with standard errors of the mean instead of the actual data points to allow reliable appreciation of the data dispersion.

    Response: We have modified our figures and have performed statistical analysis according to the suggestions of the reviewers, please see responses to comments 1-9, and 13-19.

    Comment 4: It is difficult to understand how minute changes in miR-184 levels can lead to over an order of magnitude differences (in some cases) in dilp8 mRNA levels considering that it is a stoichiometric relationship. Maybe ?miR-184-Dicer1? complexes are highly stable and re-used for multiple dilp8 transcripts - the authors could discuss how they understand this occurring in their manuscript.

    On the same line, discussion is also rather weak on what regards the mechanism of control of dilp8 mRNA levels by miR-184. Please discuss eg, the evidence for mRNA degradation induction by microRNAs with this UTR binding profile (imperfect UTR binding Fig S4) and-if appropriate-how other possible regulatory models (direct and indirect) could explain the findings.

    Response: We accept the reviewers comment that 25-30% reduction of miR-184 is low in comparison to the many fold increase in dilp8 levels. We believe that both post-transcriptional and transcriptional changes are responsible for the induction of dilp8 in response to tissue damage. However, our experiments suggest the role of post-transcriptional regulation by miR-184, as pupariation delay is rescued by miR-184 overexpression (also please see the response to the previous comment). We are not ruling out the possibility of transcriptional regulation of dilp8 mRNA, rather we are suggesting the possibility that both transcriptional and post-transcriptional means are responsible for changes in dilp8. Moreover, we have not performed absolute measurement of miR-184 in the imaginal discs (what we show is a comparison between control and RicinA expression), hence we do not have an exact estimate of how many miR-184 molecules are reduced and if they would be greatly equal or more in comparison to the dilp8 mRNA molecules that are upregulated, as again while measuring dilp8 mRNA we are not checking how many molecules of dilp8 exactly are increased. As the reviewer suggests, it is possible that miR-184-RISC could be stable to handle multiple dilp8 molecules one after the other, hence it is not a 1:1 relationship between miR-184:dilp8. We have included this in the manuscript. It is also known that imperfect 3’UTR binding as seen in most animal microRNAs leads to translational repression and mRNA deadenylation, which eventually results in mRNA degradation.

    Comment 5: ____We suggest the authors carefully revise their citations to cite appropriate work that supports the claims, and also to avoid missing the seminal studies that report the claims they cite.

    Response: We are really apologetic for the errors citing the key references. We are grateful to the reviewers for correcting this for us. We have made changes to the text to include and correct the references.

    We have the suggestions below which we hope will help the authors improve their manuscript. If the authors address these points raised above, we believe the manuscript should be a valuable contribution to the field, and help in the understanding of how tissues respond to growth aberrations and the regulation of transcript levels by microRNAs.

    Detailed Comments:

    Comment 1. Results 1st paragraph: please describe the screen in more detail. As written, one only discovers it was a miRNA loss-of-function screen when reading the legend of Table S1. Please show the original data of the screen - with dispersion if possible.

    Response: We thank the reviewers for these suggestions, we have now included the data from the screen with SEM, and p-values.

    Comment 2. Results 1st paragraph, Fourth line, "While several miRNAs caused delays in pupariation by 12 hours or more..". Please correct, as actually loss of miRNAs caused delays.

    Response: We thank the reviewer for pointing out this error, we have corrected the text accordingly.

    Comment 3. ____Results (Figure 1) - It says that data from three independent experiments are shown. However there is no dispersion in the data. Could the authors please explain this? Are the results of the three experiments summed and presented as one? or is this one of the three?

    Response: We thank the reviewers for these suggestions and have plotted data with the SEM values.

    Comment 4. It is reported in the legend of Figure S2 that LogRank test was performed to determine statistical significance. However, no statistical data is presented. Please show the results.

    __Response: __We thank the reviewers for these suggestions to improve the data presentation, we have incorporated the p-value as suggested.

    Comment 5. Fig2A and B. Please show the data points in the bar graphs (as in Figure. 2C), or choose another data representation. ____Please consider redoing statistical analysis with a simple t-test. ____It is not clear to me why ANOVA was used to compare two samples. Please state that data are normalized also to control (tub-GAL4>UAS-scramble). Please ____state____ the h post-hatching from which the RNA samples were collected (as in Fig 2C for 20HE quantification).

    __Response: __We thank the reviewers for these suggestions to improve the data presentation, we have incorporated all changes as suggested. Similar changes have been incorporated to the rest of the figures of the manuscript as well. Hours post-hatching information for each figure is now added to the figure legends. __ __

    Comment 6. Fig2C. Fig legend states the bar graphs are "absolute values". Please specify if the bar represents the average, median or something else.

    Response: We thank the reviewer for pointing this out, we have made the suggested changes.

    Comment 7. Throughout the manuscript: please use GAL4 in capital letters or at least standardize it throughout the ms. Currently there are GAL4s and Gal4s.. eg compare Fig 2 and 3 legends.

    Response: We thank the reviewer for pointing this out, we have incorporated all changes as recommended.

    Comment 8. FigS3A and B. Please revise as Fig2A and B above. and apply the same criteria in the respective figure legend.

    __Response: __We thank the reviewer for pointing this out, we have made the changes as recommended.

    Comment 9. Fig. 4 - please indicate on the figures what is whole larvae and what is wing imaginal discs. This will facilitate understanding of the figure.

    __Response: __We thank the reviewers for these suggestions and have included this information in all the figures.

    Comment 10. Fig 4 - Data - Authors do not show that rn-GAL4>miR-184-sponge causes up regulation of dilp8 mRNA levels, hence the model is weakened. Doing this experiment would significantly strengthen the study whatever the result is.

    Response: We thank the reviewer for pointing this out and we have included this in the manuscript (Fig S5B).

    Comment 11. The dilp8-3'UTR experiment is weak especially because its generation is not sufficiently well described in the manuscript. "The dilp8 3'UTR-GFP reporter line was created as described in (Vargheese & Cohen, 2007)" is not sufficient. Please describe the construct generation in sufficient detail so that the experiments can be reproduced by others.

    Response: We thank the reviewer for pointing this out and we have elaborated in the methods section on how we generated the dilp8 3'UTR-GFP reporter and dilp8 3'UTR mutant GFP reporter lines. The plasmid was originally created in Steve Cohen’s lab at EMBL, by modifying pCasper4 plasmid, by introducing a tubulin promoter, EGFP and a multiple cloning site, which allows one to clone 3’UTRs of target genes into this plasmid. Not1 and Xho1 sites were used to clone the dilp8-3’UTR and mut-3’UTR. We hope this explains our strategy sufficiently.

    Comment 12. Making assumptions, if the construct is as described in Vargheese & Cohen, 2007 and contains all of the dilp8 3'UTR - it should be a Tubulin-driven GFP gene with a dilp8-3'UTR "Tub-GFP-(dilp8 3'UTR)". In this case the authors need to rule out the alternative interpretation of the result in Fig. 4D by showing that the expression of miR-184 does not down regulate Tub-GFP expression itself. The best scenario would be to have a mutated dilp8 3'UTR for the miR-184 recognition site. This experiment would significantly strengthen the study and model.

    Response: We thank the reviewer for pointing this out. We agree with the reviewers that this experiment is needed to prove direct regulation of the dilp8-3’UTR by miR-184. We have mutated the sequences complementary to the seed region of miR-184 in the dilp8-3’UTR, and demonstrated that overexpression of miR-184 does not regulate the mutated tub-GFP-(dilp8 3'UTR) expression. This confirms that the dilp8 gene is a direct target of miR-184. This data is added to the manuscript as Figs 4E-F’.

    Comment 13. Figure 4C-D please separate dilp8 from 3'UTR with a space or hyphen.

    Response: We thank the reviewer for pointing this out and have separated dilp8 from 3’UTR with a hyphen.

    Comment 14. Figure 4E. Please name the dilp8 allele as MI00727 as it is not a KO, but rather a hypomorphic mutation (fully WT dilp8 transcripts are still generated, albeit at a much lower level).

    Response: We thank the reviewer for pointing this out and we have made the necessary changes.

    Comment ____15. Figure 6D: please add UAS to bskDN/+. All figures have rn-GAL4 alone or with UAS-GFP as control. This finding would be strengthened with this other control, especially because the size effect is small.____ This being said a general comment for all experiments is that hemi-controls are generally missing for all figures. eg, in Fig 3. One would typically include controls such as A. Phm>+ and +>miR.184; B. aug21>+ and +>miR.184; C. ptth>+ and +>miR.184; D. rn>+ and +>miR.184

    Response: We thank the reviewer for pointing this out. We have added UAS to bskDN, now Fig 5D and have also added the rnGAL4/+ control. We have also performed various hemi-control experiments as suggested by the reviewer to our best capabilities. We have added a separate graph with the hemicontrols in the as a Reviewer Response Figure 1.

    Comment 16. Figure 7: Are IPCs necessary for the model? If not, I suggest removing them and placing the Lgr3 neuron cell bodies much more anterior in this scheme. Their cell bodies are as anterior and rostral as it gets, approximately where the IPCs are depicted in this type of view of the CNS.

    Response: We thank the reviewer for pointing this out and have removed IPCs from the figure, this figure is now labelled as Fig. 6.

    Comment ____17. Table S1- It would be preferable to see the data of these experiments, but if the authors prefer to show this data in a table, please at least add the dispersion analyses (eg standard deviation.. OR median+-quartiles OR Confidence intervals..), N of animals analysed, and statistics against controls.

    Response: We thank the reviewer for pointing this out, we have added the number of larvae analysed, SEM values and statistics against the control condition.

    Comment ____18. In all figures with pupariation time: please also indicate significant findings in the graphs (with an asterisk, for instance) and adjust figure legends accordingly. This could facilitate understanding the data.

    __Response: __Thanks for the suggestion. We have incorporated this information into figure legends.

    Comment ____19. Please revise Figure legends for punctuation.

    __Response: __We have rectified all the errors in punctuation. We thank the reviewers for suggesting this.

    __Comment ____20. __

    a) Abstract:

    Line 10: What is the evidence to call Dilp8 a "paracrine" factor?

    Response: We thank the reviewer for pointing this out, we have changed the text to ‘secreted factor’.

    b) Introduction:

    4th paragraph, 3rd sentence " Dilp8... buffers developmental noise and delays pupariation..." Buffering of developmental noise was first shown in Garelli et al., Science 2012, so this publication should be cited. ____4th paragraph, 5th sentence: please include Jaszczak et al., Genetics 2016. This paper was published together with the 2015 papers, just a matter of timing that it got a 2016 date. Moreover, I do not think Katsuyama et al., 2015 is well cited to back up the statement in this sentence, hence I recommend removing that citation in this sentence.

    Response: We thank the reviewer for pointing this out and have made necessary changes.

    c) 6th paragraph: 5th line "targeting dilp8" : please specify if you mean the gene or the mRNA, or both. Same for line 7.

    Response: We thank the reviewer for pointing this out and have made necessary changes.

    d) Results Page 10, 1st paragraph, 1st sentence: the works cited are not the appropriate studies that demonstrated what is being stated. This was shown in Garelli et al., Science 2012 and Colombani et al., Science 2012. Results Page 10, 1st paragraph, line 11: Please also cite Colombani et al., Science 2012, who first showed that JNK is required for dilp8 regulation.

    Response: We thank the reviewer for pointing this out and are extremely apologetic for this oversight. We have made necessary changes to the manuscript.

    e) Discussion, 2nd paragraph, line 4: again, please indicate the rationale for using "paracrine" to describe Dilp8's activities. The current widely accepted model is that Dilp8 acts on interneurons in the brain ____(eg, reviewed in Juarez-Carreno et al., Cell Stress, 2018; Gontijo and Garelli, Mech Dev, 2018; Mirth and Shingleton, Front Cell Dev Biol, 2019; Texada et al., Genetics 2020; Boulan and Leopold, 2021).____ In order to reach the brain, Dilp8 has to be secreted from the discs and travel to the brain. This is as an endocrine mechanism as it gets for a small larva, considering that some discs can be on the opposite side of the larva (eg, genital discs). While this does not exclude that Dilp8 could also act paracrinally, the only evidence that I am aware of comes from other contexts such as during transdetermination (where Dilp8 has been proposed to work in an autocrine or paracrine fashion, via Drl in imaginal discs (Nemoto et al., Genes to Cells, 2023), however, this is not cited appropriately in this manuscript and is less related to the Lgr3-dependent pathway being studied here.

    Response: We totally agree with the reviewer and appreciate clarifying this for us. We have made necessary changes to the text.

    f) Discussion Page 13, 1st paragraph, This claim is supported by data presented in Garelli et al., Science 2012, not the other two papers. Garelli et al., 2015 shows that the Lgr3 receptor also participates in buffering developmental noise. Other studies have corroborated the Garelli et al., 2012 finding: eg, Colombani et al., Curr Biol 2015; Boone et al., Nat Commun 2016; Blanco-Obregon et al., Nat Commun 2022). Many other studies have shown that Dilp8 promotes developmental stability under tissue stress and challenges.

    Discussion Page 12, 3rd paragraph, 2nd sentence: "The Lgr3 neurons directly interact with ... PTTH ...and insulin-producing neurons" Please cite Colombani et al., 2015 and Vallejo et al., Science 2015. Vallejo et al., propose that circuit with insulin-producing neurons. In the 3rd sentence, only Jaszczak et al., 2016 is cited, whereas this claim/model comes from many studies, such as Halme et al., Curr Biol, 2010; Hackney et al., PLoS One 2012; Garelli et al. Science 2012; Colombani et al., Science, 2012; and the Lgr3 papers from 2015). Jaszczak et al., actually propose that Lgr3 is also required in the ring gland in addition to neurons.

    Discussion page 14 last paragraph,10 line, "In Aedes aegypti ....regulates ilp8 (Ling et al., 2017)". As far as I understand mosquitoes do not have a dilp8 orthologue (see for instance Gontijo and Gontijo, Mech Dev 2018; and Jan Veenstra's work). ilp nomenclature (numbering) does not follow that of Drosophila, so ilp8 is probably a typical Insulin/IGF-like peptide and is NOT an orthologue of Dilp8, a relaxin, so this citation needs to be removed or placed into the broader context of microRNA regulation of ilps.

    Response: We are really sorry for the numerous glaring errors in the references. We thank the reviewers for correcting this for us. We have made necessary changes to the text.

    Thank you for the opportunity to review your interesting work,

    Alisson Gontijo and Rebeca Zanini

    Reviewer #3 (Significance (Required)):

    If the authors address these points raised above, we believe the manuscript should be a valuable contribution to the field, and help in the understanding of how tissues respond to growth aberrations and the regulation of transcript levels by microRNAs.

    __Author’s concluding response: __

    We thank all the reviewers for the overall positive comments and suggestions that we believe have helped us to improve our manuscript. We have incorporated all the changes suggested, especially regarding errors in citing key references. We have performed most of the experimental suggestions. Also, we have modified the way in which graphs are presented, including statistical tests as suggested by the reviewers. Several controls have been performed to strengthen the manuscript further. We believe that this review process aided in significantly improving this manuscript.

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    Referee #3

    Evidence, reproducibility and clarity

    This is an interesting study demonstrating an interaction between miR-184 and the Drosophila insulin-like peptide 8 (dilp8) in the tissue damage response. The authors show that Dilp8 activity is negatively regulated by miR-184, apparently through direct interaction between miR-184 and the dilp8-3'UTR, which leads to lower dilp8 mRNA transcript levels, via an undetermined mechanism, supposedly its degradation? Furthermore, the authors show that during aberrant tissue growth, miR-184 levels are very slightly downregulated (see comment below), and based on other experiments, imply causation of this with the increased dilp8 mRNA levels that occur in these tissues, again via an unclear mechanism: upregulation or stabilization of dilp8 mRNA. The authors present evidence that the JNK pathway, which had been known to be critical for dilp8 mRNA upregulation upon tissue damage, does so via miR-184. The data showing the direct regulation of dilp8-3'UTR by miR-184 are not very strong and would require more controls to strengthen the claim, as described below. The miR-184 effects are also very small (less than 2-fold reduction with tissue damage; or less than 2-fold induction with JNK-pathway inhibition via bsk-DN). These two points are the weakest part of the manuscript and model. Regarding the expression levels, it does not help that the authors show bar graphs with standard errors of the mean instead of the actual datapoints to allow reliable appreciation of the data dispersion. It is difficult to understand how minute changes in miR-184 levels can lead to over an order of magnitude differences (in some cases) in dilp8 mRNA levels considering that it is a stoichiometric relationship. Maybe ?miR-184-Dicer1? complexes are highly stable and re-used for multiple dilp8 transcripts - the authors could discuss how they understand this occurring in their manuscript. On the same line, discussion is also rather weak on what regards the mechanism of control of dilp8 mRNA levels by miR-184. Please discuss eg, the evidence for mRNA degradation induction by microRNAs with this UTR binding profile (imperfect UTR binding Fig S4) and-if appropriate-how other possible regulatory models (direct and indirect) could explain the findings. We suggest the authors carefully revise their citations to cite appropriate work that supports the claims, and also to avoid missing the seminal studies that report the claims they cite. We have the suggestions below which we hope will help the authors improve their manuscript. If the authors address these points raised above, we believe the manuscript should be a valuable contribution to the field, and help in the understanding of how tissues respond to growth aberrations and the regulation of transcript levels by microRNAs.

    Comments:

    Results 1st paragraph: please describe the screen in more detail. As written, one only discovers it was a miRNA loss-of-function screen when reading the legend of Table S1. Please show the original data of the screen - with dispersion if possible.

    Results 1st paragraph, Fourth line, "While several miRNAs caused delays in pupariation by 12 hours or more..". Please correct, as actually loss of miRNAs caused delays.

    Results (Figure 1) - It says that data from three independent experiments are shown. However there is no dispersion in the data. Could the authors please explain this? Are the results of the three experiments summed and presented as one? or is this one of the three?

    It is reported in the legend of Figure S2 that LogRank test was performed to determine statistical significance. However, no statistical data is presented. Please show the results.

    Fig2A and B. Please show the data points in the bar graphs (as in Figure. 2C), or choose another data representation. Please consider redoing statistical analysis with a simple t-test. It is not clear to me why ANOVA was used to compare two samples. Please state that data are normalized also to control (tub-GAL4>UAS-scramble). Please state the h post-hatching from which the RNA samples were collected (as in Fig 2C for 20HE quantification).

    Fig2C. Fig legend states the bar graphs are "absolute values". Please specify if the bar represents the average, median or something else.

    Throughout the manuscript: please use GAL4 in capital letters or at least standardize it throughout the ms. Currently there are GAL4s and Gal4s.. eg compare Fig 2 and 3 legends.

    FigS3A and B. Please revise as Fig2A and B above. and apply the same criteria in the respective figure legend.

    Fig. 4 - please indicate on the figures what is whole larvae and what is wing imaginal discs. This will facilitate understanding of the figure.

    Fig 4 - Data - Authors do not show that rn-GAL4>miR-184-sponge causes up regulation of dilp8 mRNA levels, hence the model is weakened. Doing this experiment would significantly strengthen the study whatever the result is.

    The dilp8-3'UTR experiment is weak especially because its generation is not sufficiently well described in the manuscript. "The dilp8 3'UTR-GFP reporter line was created as described in (Vargheese & Cohen, 2007)" is not sufficient. Please describe the construct generation in sufficient detail so that the experiments can be reproduced by others.

    Making assumptions, if the construct is as described in Vargheese & Cohen, 2007 and contains all of the dilp8 3'UTR - it should be a Tubulin-driven GFP gene with a dilp8-3'UTR "Tub-GFP-(dilp8 3'UTR)". In this case the authors need to rule out the alternative interpretation of the result in Fig. 4D by showing that the expression of miR-184 does not down regulate Tub-GFP expression itself. The best scenario would be to have a mutated dilp8 3'UTR for the miR-184 recognition site. This experiment would significantly strengthen the study and model.

    Figure 4C-D please separate dilp8 from 3'UTR with a space or hyphen.

    Figure 4E. Please name the dilp8 allele as MI00727 as it is not a KO, but rather a hypomorphic mutation (fully WT dilp8 transcripts are still generated, albeit at a much lower level).

    Figure 6D: please add UAS to bskDN/+. All figures have rn-GAL4 alone or with UAS-GFP as control. This finding would be strengthened with this other control, especially because the size effect is small. This being said a general comment for all experiments is that hemi-controls are generally missing for all figures. eg, in Fig 3. One would typically include controls such as A. Phm>+ and +>miR.184; B. aug21>+ and +>miR.184; C. ptth>+ and +>miR.184; D. rn>+ and +>miR.184

    Figure 7: Are IPCs necessary for the model? If not, I suggest removing them and placing the Lgr3 neuron cell bodies much more anterior in this scheme. Their cell bodies are as anterior and rostral as it gets, approximately where the IPCs are depicted in this type of view of the CNS.

    Table S1- It would be preferable to see the data of these experiments, but if the authors prefer to show this data in a table, please at least add the dispersion analyses (eg standard deviation.. OR median+-quartiles OR Confidence intervals..), N of animals analysed, and statistics against controls.

    In all figures with pupariation time: please also indicate significant findings in the graphs (with an asterisk, for instance) and adjust figure legends accordingly. This could facilitate understanding the data.

    Please revise Figure legends for punctuation.

    Abstract: Line 10: What is the evidence to call Dilp8 a "paracrine" factor?

    Introduction:

    4th paragraph, 3rd sentence " Dilp8... buffers developmental noise and delays pupariation..." Buffering of developmental noise was first shown in Garelli et al., Science 2012, so this publication should be cited.

    4th paragraph, 5th sentence: please include Jaszczak et al., Genetics 2016. This paper was published together with the 2015 papers, just a mater of timing that it got a 2016 date. Moreover, I do not think Katsuyama et al., 2015 is well cited to back up the statement in this sentence, hence I recommend removing that citation in this sentence.

    6th paragraph: 5th line "targeting dilp8" : please specify if you mean the gene or the mRNA, or both. Same for line 7.

    Results Page 10, 1st paragraph, 1st sentence: the works cited are not the appropriate studies that demonstrated what is being stated. This was shown in Garelli et al., Science 2012 and Colombani et al., Science 2012.

    Results Page 10, 1st pagragraph, line 11: Please also cite Colombani et al., Science 2012, who first showed that JNK is required for dilp8 regulation.

    Discussion, 2nd paragraph, line 4: again, please indicate the rationale for using "paracrine" to describe Dilp8's activities. The current widely accepted model is that Dilp8 acts on interneurons in the brain (eg, reviewed in Juarez-Carreno et al., Cell Stress, 2018; Gontijo and Garelli, Mech Dev, 2018; Mirth and Shingleton, Front Cell Dev Biol, 2019; Texada et al., Genetics 2020; Boulan and Leopold, 2021). In order to reach the brain, Dilp8 has to be secreted from the discs and travel to the brain. This is as an endocrine mechanism as it gets for a small larva, considering that some discs can be in the opposite side of the larva (eg, genital discs). While this does not exclude that Dilp8 could also act paracrinally, the only evidence that I am aware of comes from other contexts such as during transdetermination (where Dilp8 has been proposed to work in an autocrine or paracrine fashion, via Drl in imaginal discs (Nemoto et al., Genes to Cells, 2023), however, this is not cited appropriately in this manuscript and is less related to the Lgr3-dependent pathway being studied here.

    Discussion Page 13, 1st paragraph, This claim is supported by data presented in Garelli et al., Science 2012, not the other two papers. Garelli et al., 2015 shows that the Lgr3 receptor also participates in buffering developmental noise. Other studies have corroborated the Garelli et al., 2012 finding: eg, Colombani et al., Curr Biol 2015; Boone et al., Nat Commun 2016; Blanco-Obregon et al., Nat Commun 2022). Many other studies have shown that Dilp8 promotes developmental stability under tissue stress and challenges.

    Discussion Page 12, 3rd paragraph, 2nd sentence: "The Lgr3 neurons directly interact with ... PTTH ...and insulin-producing neurons" Please cite Colombani et al., 2015 and Vallejo et al., Science 2015. Vallejo et al., propose that circuit with insulin-producing neurons. In the 3rd sentence, only Jaszczak et al., 2016 is cited, whereas this claim/model comes from many studies, such as Halme et al., Curr Biol, 2010; Hackney et al., PLoS One 2012; Garelli et al. Science 2012; Colombani et al., Science, 2012; and the Lgr3 papers from 2015). Jaszczak et al., actually propose that Lgr3 is also required in the ring gland in addition to neurons.

    Discussion page 14 last paragraph,10 line, "In Aedes aegypti ....regulates ilp8 (Ling et al., 2017)". As far as I understand mosquitoes do not have a dilp8 orthologue (see for instance Gontijo and Gontijo, Mech Dev 2018; and Jan Veenstra's work). ilp nomenclature (numbering) does not follow that of Drosophila, so ilp8 is probably a typical Insulin/IGF-like peptide and is NOT an orthologue of Dilp8, a relaxin, so this citation needs to be removed or placed into the broader context of microRNA regulation of ilps.

    Thank you for the opportunity to review your interesting work, Alisson Gontijo and Rebeca Zanini

    Significance

    If the authors address these points raised above, we believe the manuscript should be a valuable contribution to the field, and help in the understanding of how tissues respond to growth aberrations and the regulation of transcript levels by microRNAs.

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    Referee #2

    Evidence, reproducibility and clarity

    Drosophila has helped to characterize the mechanisms that coordinate tissue growth with developmental timing. The insulin/relaxin-like peptide Dilp8 has been identified as a key factor that communicates the abnormal growth status of larval imaginal discs to neuroendocrine neurons responsible for regulating the timing of metamorphosis. Dilp8, derived from imaginal discs, targets four Lgr3-positive neurons in the central nervous system, activating cyclic-AMP signaling in an Lgr3-dependent manner. This signaling pathway reduces the production of the molting hormone, ecdysone, delaying the onset of metamorphosis. Simultaneously, the growth rates of healthy imaginal tissues slow down, enabling the development of proportionate individuals. In this manuscript "miR-184 modulates dilp8 to control developmental timing during normal growth conditions and in response to developmental perturbations" by Dr. Varghese and colleagues, the authors identify a new post transcriptional regulator of Dilp8. The authors show that miR-184 plays a pivotal role in tissue damage responses by inducing dilp8 expression, which in turn delays pupariation to allow sufficient time for damage repair mechanisms to take effect.

    Major points:

    • In most of the experiments for percentage of pupariation, the 50% pupariation in control is around 110 hours AED in figures 1, 2 and 3. In figures 5 and 6 using the UAS Ricin, the controls are more around 90 hours AED. Why this discrepancy?
    • What is the mechanism behind the expression of miR-184 in stress conditions? Does miR-184 also implicated in other conditions giving rise to a developmental delay (X-rays irradiation or animal bearing rasV12, scrib-/- tumors)?
    • dilp8 mutant animals have also been shown to be more resistant to starvation or desiccation (https://doi.org/10.3389/fendo.2020.00461 ). Is miR-184 implicated in this answer?
    • dilp8 expression has been also shown to be regulated by Xrp1 in response to ribosome stress (https://doi.org/10.1016/j.devcel.2019.03.016). This paper should be included in the manuscript Is it possible that the expression levels of miR184 are regulated by Xrp1?

    Minor points:

    • Does the overexpression of miR184 induce an increased fluctuating asymmetry?
    • There are 2 references Colombani et al. (2012 for Dilp8 and 2015 for Lgr3). Can you double check that they are used accordingly

    Significance

    Altogether, the paper present compiling lines of evidence supporting the proposed model. The experiments are well designed and are convincing. The papers is interesting and relevant for a broad audience.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary

    In this study, Fernandes and colleagues addressed the question of the role of micro-RNAs in regulating the coupling between organ growth and developmental timing. Using Drosophila, they identified the conserved micro-RNA miR-184 as a regulator of the developmental transition between juvenile larval stages and metamorphosis. This transition is under the control of the steroid hormone Ecdysone, and has been shown to be modulated in case of abnormal tissue growth to adjust the duration of larval growth in response to developmental perturbations. The relaxin-like hormone Dilp8 has been identified as a key secreted factor involved in this coupling. Here, the authors show that miR-184 is involved in the regulation of Dilp8 expression both in physiological conditions and upon growth perturbation. They propose that this function is carried out in imaginal tissues, where miR-184 levels are modulated by tissue stress. While several factors have already been involved in triggering sharp dilp8 induction at the transcriptional level, this study adds another level of complexity to the regulation of Dilp8 by proposing that its expression is fine-tunned post-transcriptionally through repression by miR-184.

    Major Comments

    Overall, the manuscript is well organized, and the logics of the experimental plan well presented. The results are clear, and I appreciate the quality of the pupariation curves. However, I believe that two main conclusions of the paper are not fully supported by the results presented in the figures: the direct regulation of dilp8 3'UTR by miR-184, and the specificity of this regulation in imaginal discs. Here I develop in more details these two aspects.

    1. The strategy of the 3'UTR sensor is not fully optimized. Indeed, in most experiments, qRT-PCR is used to assess dilp8 expression levels, although it reflects both transcriptional and post-transcriptional. Importantly, to show that post-transcriptional regulation is involved in the response to tissue damage, the levels of the 3'UTR sensor should be analyzed in discs expressing RAcs (showing at the same time that the response is cell-autonomous in the discs). The expected upregulation of the sensor should be prevented by simultaneous expression of miR-184. This approach would shed light on the relative contribution of transcriptional versus post-transcriptional regulation of dilp8 in response to growth perturbation.
    2. In my opinion, the use of a 3'UTR sensor is not sufficient to conclude that the regulation by miR-184 is direct, as miR-184 could also regulate an intermediate factor that acts on dilp8 post-transcriptional regulation. To solve this issue, a common strategy is to generate a 3'UTR sensor with mutated binding sites that should abolish the regulation by miR-184. This mutated 3'UTR might also respond differently to tissue damage, which would strongly support the conclusions of the study.
    3. Concerning the tissue-specific regulation of Dilp8 by miR-184, these results need to be strengthened. Indeed, this comes mostly from phenotypes observed with rn-GAL4. Although this is a classical tool for driving expression in imaginal discs, rn-GAL4 also drives strong expression in other tissues that could contribute to triggering a delay, such as the CNS and part of the gut (proventriculus). In our hands, some growth phenotypes in the wing obtained with rn-GAL4 could be fully reverted by blocking GAL4 in the CNS indicating that the phenotype was not wing-specific. Importantly, miR-184 seems to be highly expressed in the CNS according to FlyBase, reinforcing the possibility that it plays a role in this organ. Here I propose approaches to confirm that miR-184 mediated regulation of dilp8 and developmental timing indeed occur in the discs:
      • Another driver with less secondary expression sites could be used (pdmR11F02-GAL4), or rn-GAL4 could be combined with an elav-GAL80 to prevent expression in most neurons.
      • The authors could identify the source of Dilp8 upregulation in miR-184 mutants using tissue-specific qRT-PCR instead of whole larvae expression like in Fig 4A-B.
      • This tissue-specific upregulation could be functionally tested using a rescue experiment, in which the delay observed in miR-184 mutants could be rescued by disc-specific downregulation of Dilp8 (using pdm2-GAL4 for instance).

    Optional: Because it is known that dilp8 is strongly regulated at the transcriptional level, the relative input from post-transcriptional upregulation is an important question arising from this study. Although it might be a more long-term approach, I believe that generating a Dilp8 mutant lacking its 3'UTR or, even better, with mutated miR-184 binding sites, would shed light on the role of this regulation for the response to growth perturbation and/or developmental stability (fluctuating asymmetry).

    Minor Comments

    • I think that a number of results could be moved to SI as they are either controls, or reproduce published data without bringing novelty. For instance, results in Fig 5A-D are similar to data published by Sanchez et al, as stated in the text. Fig6A as well.
    • Fig 6D is quite mysterious, as it suggests that basal JNK activation regulates miR-184, which is different from a context of tissue damage. I think that this result could be removed. Alternatively, if the authors want to dig in that direction, more experiments should be provided, such as bskDN expression in an RAcs context and the effects on miR-184 levels and the 3'UTR sensor (since transcript levels are already published).
    • The references related to Dilp8 should be checked more in detail in the intro and discussion. About Dilp8 and developmental stability: remove the ref to Colombani et al 2012, instead put Boone et al 2016 and add Blanco-Obregon et al 2022 (in addition to Garelli et al 2012 who initially identified this phenotype. About Lgr3 as the receptor for Dilp8: add Colombani et al, Current Biology 2015, and cite here Vallejo et al 2015, Garelli et al 2015. Among the important transcriptional regulators of Dilp8, Xrp1 could be mentioned (Boulan et al 2019, Destefanis et al 2022) as it plays a complementary function to JNK depending on the type of tissue stress.

    Significance

    General Assessment

    This study provides convincing data showing that the conserved microRNA miR-184 plays a role in regulating developmental timing in Drosophila through modulating the levels of Dilp8, a key factor in the coupling between tissue growth and developmental transitions. The results are convincing, but the general conclusions of the paper need to be strengthened regarding the direct regulation of dilp8 by miR-184 and the tissue-specificity of this interaction.

    Advance

    Dilp8 is a key factor that modulates growth and timing in response to developmental perturbations and contributes to developmental precision in physiological conditions. As such, its regulation has been studied by different groups in the last decade, leading to the identification of several inputs for its transcriptional regulation. Here, the authors uncover a post-transcriptional regulation by miR-184, adding another level of regulation of Dilp8 that contribute to ensuring proper regulation of developmental timing, and opening the possibility that miR-184 might play similar roles in other species.

    Audience

    This study is of interest for researchers in the field of basic science, with a focus on developmental timing, tissue damage and biological function of microRNAs.

    Reviewer expertise

    Drosophila, growth control, developmental timing, Dilp8.

  5. Version published to 10.1101/2024.07.08.602443 on bioRxiv