ROS-mediated TNFR Wengen activation in response to apoptosis

  1. José Esteban-Collado
  2. Mar Fernàndez-Mañas
  3. Manuel Fernández-Moreno
  4. Ignacio Maeso
  5. Montserrat Corominas
  6. Florenci Serras

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Abstract

The activation of tumor necrosis factor receptors (TNFR) controls pleiotropic pro-inflammatory functions ranging from apoptosis to survival. The ability to trigger a particular function will depend on the upstream activation, association with regulatory complexes and downstream pathways. In Drosophila, two TNFRs have been identified, Wengen (Wgn) and Grindelwald (Grnd). Although several reports associate these receptors with JNK-dependent apoptosis, it has recently been found that Wgn activates a variety of functions. We demonstrate that Wgn is required for survival by protecting cells from apoptosis. This is mediated by the signaling molecule dTRAF1 and results in the activation of the p38 MAP kinase signaling pathway. Remarkably, Wgn is required for apoptosis-induced regeneration and is activated by the reactive oxygen species (ROS) produced following apoptosis. This ROS activation is exclusive for Wgn, but not for Grnd, and occurs in the absence of the ligand Eiger/TNFα. Furthermore, based on protein sequence conservation, the extracellular Cys-rich domain of Grnd is much more divergent and phylogenetically restricted than that of Wgn, which is more similar to TNFR families from other animals, including those of human TNFRs. Taken together, our results show a novel function for a TNFR that responds to cellular damage by ensuring the cell survival required for the response to damage.

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    Reply to the reviewers

    1. General Statements [optional]

    All four reviewers have positive comments on the paper. We totally agree with their comments, and proposed controls and experiments. Most of them are already introduced in the present text and several new figures added, as we had the controls/experiments proposed. Few others are now being done and we hope to have the complete set of experiments ready in 2-3 months.

    2. Description of the planned revisions

    Insert here a point-by-point reply that explains what revisions, additional experimentations and analyses are planned to address the points raised by the referees.

    Reviewer #1

    Most comments of this reviewer have already been done and included in the transferred manuscript, except for part of the first comment:

    1.1 b. Is it possible that the loss of function of Wengen on its own has a phenotype? If so, that would suggest that Wgn in addition to its role in regeneration might be implicated in pro-survival processes in homeostatic conditions?

    This issue is very important to understand the differential role of Wgn and Grnd. First of all, Wengen knock out (wgnKO; Andersen et al., 2015) is viable in homozygosis. However, in this paper we have focused on inducible mutants. Therefore, we have now crossed the flies to get the genotype hh-Gal UAS RNAi wgn and we will check for apoptotic phenotype, as suggested. This will take us few weeks of work.

    Reviewer #2 Most comments have been already carried out and included in the transferred manuscript, except these ones:

    *2.3. Aside from wgn, other RNAi experiments are not validated through independent RNAi lines. I suggest expanding the Supplemental Figures to reproduce a few key findings with independent RNAi lines. *

    We have recently received a set of independent RNAi line to repeat the experiments for Traf1, Traf2, Ask1 from Bloomington Stock Center. And We did not do it before mainly because we wanted to focus on wgn and grnd. However, we agree with the Reviewer 2 and we will do the experiments. Another RNAi from VDRC for grnd and Tak1 have been ordered. These experiments will take about 2 months from the crosses to the analysis of results (some flies still to arrive, and many crosses will be done at 17ºC).

    *2 4. In Figure 1E, the authors show that wgn RNAi enhances cell death caused by hh>egr. What is missing here is a wgn RNAi control without hh>egr. Is there any cell death caused by the loss of wgn alone (without hh>egr)? *

    This control is now in progress. Expected to have it complete in 2 weeks.

    *2.5. If wgn RNAi causes some degree of cell death, is the observed effect with hh>egr a significant genetic interaction, or merely additive? *

    The result from the previous comment will help us to respond this point.

      1. Is the wgn-p38 pathway sufficient to block egr induced cell death? The authors could test this by having hh>egr in the licT1.1 background. The authors have a more complex experiment in Figure 3, where licT1.1 is introduced into the hh>egr, wgn RNAi background. However, testing the effect of licT1.1 without wgn would establish a more direct relationship between egr and wgn-p38. *

    We have set the crosses for the experiment hh>egr and licT1.1 as suggested. The results will be included in the new version of the manuscript. 1 month.

    Reviewer #3

    All comments already carried out and included in the transferred manuscript. See next sections.

    __Reviewer #4 __

    Major comments:

    *4.3 In Figure 5, the cells expressing Rpr appeared to be pulled/extruded basally as expected. It would be beneficial to quantify Wgn and Grnd signals along cross-sections and provide higher magnification images of domain boundaries to illustrate differences in TNFR localization and levels. ** The micrographs for Grnd Figure 5B,D, F capture substantial signal from the peripodial epithelium where the salE/Pv> driver is likely not active? *

    We will do a thorough quantification of high-resolution stacks of images and include higher magnification of the analyzed stacks. To this aim, we need some more weeks to collect the images of each genotype, processed and quantify them. We propose to do have this work done in two months.

    *4.4 The non-autonomous induction of Wgn seems stronger when facing dying Rpr overexpressing cells simultaneously depleted of Eiger compared to Rpr OE alone. Should this be a reproducible, could the authors discuss potential reason for this observation? *

    It is difficult to respond this question, without quantification. The quantification suggested in the previous point, will allow us to state if Wgn is more accumulated in rpr +egr than *rpr *alone. Therefore, the previous point will tell us if there are significant differences and if, so it will help us to discuss it.

    Timing: The entire plan can be executed in 2-3 month.

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

    Please insert a point-by-point reply describing the revisions that were already carried out and included in the transferred manuscript. If no revisions have been carried out yet, please leave this section empty.

    __Reviewer #1 __

    1.1 a- *The result in Fig1.H is somehow surprising. How does the overexpression of Egr induce caspase activation in the absence of its receptor Grnd? *

    The results of Fig. 1H, in which egr+grndRNAi+wgnRNAi results in high apoptosis indicates that wgn down regulation compromises survival even in the absence of grnd. The reviewer correctly points that “How does the overexpression of Egr induce caspase activation in the absence of its receptor Grnd?”.

    There is evidence that Eiger is involved in the regulation of the pro-apoptotic gene head involution defective (hid) in primordial germ cells (Maezawa 2009 Dev. Growth Differ., 51 (4) (2009), pp. 453-461) and in the elimination of damaged neurons during development (Shklover et al., 2015). Moreover, Eiger is necessary for HID stabilization and regulates HID-induced apoptosis independently of JNK signaling (Shklover et al., 2015). Therefore, in our discs *egr *activation in the absence of *grnd *and wgn can still result in apoptosis because of the absence of wgn’s survival signal and, presumably, activation of hid.

    We have introduced this issue in the text as:

    “To check for epistasis between grnd and wgn, we activated hh> egrweak and knocked down both TNFRs. We found high levels of cell death compared to wgn RNAi alone (Fig. 1H and 1I), which demonstrates that wgn down-regulation is dominant over grnd. This is surprising as it is generally assumed that Egr interacts with Grnd to induce apoptosis via JNK, which in turn activates the proapoptotic gene hid (Andersen et al., 2015; Diwanji & Bergmann, 2020; Fogarty et al., 2016; Igaki et al., 2002; Moreno, Yan, et al., 2002; Sanchez et al., 2019; Shlevkov & Morata, 2012). Interestingly, Egr is necessary for HID stabilization and can regulate HID-induced apoptosis independently of JNK (Shklover et al., 2015). Therefore, cells egrweak that downregulate grnd and wgn can still be eliminated because the lack of both Wgn-survival signal and the pro-apoptotic Grnd/JNK signal could result in an alternative pathway of apoptosis.”

    *1.2- In Fig.6, it would be relevant to include wengen inactivation within the domain where rpr is expressed to show that wengen is not required autonomously for regeneration (sal>rpr + wgn RNAi). What is the phenotype of the adult wing of sal-lexA>rpr + nub-gal4 >wgn RNAi animals.? *

    We have already added a new figure (Fig. S4C) containing this data. As shown, both *wgnRNAi *alone and wgn RNAi + rpr do not show relevant anomalies and regenerate normally. Therefore, we conclude that *wgn *is not autonomously required for regeneration.

    The adult wings sal-lexA>rpr + nub-gal4 >wgn RNA result in a strong aberration, as regeneration is inhibited. This experiment has been also added in another figure (Fig. S4B) it is done.

    *1.4 Minor- In fig.1I, it is surprising that knockdown of neither Grnd nor dTRAF2 significantly affects Egr-induced apoptosis *

    After applying a One-Way ANOVA test to compare all the groups against all the groups in fig. 1B no significative differences were detected between Control and RNAi grnd or RNAi dTRAF2 (p>0,05). But if we apply a Student’s T test, which is less restrictive, we obtain, indeed, significative differences:

    Control vs. RNAigrnd p=9,48x10-7

    Control vs. RNAi dTRAF2 p=2,47x10-7

    We have now added in the text:

    “Note that when egrweak cells downregulated dTRAF2 or grnd the cell death area ratio is slightly lower than egrweak alone (Fig. 1I), comfirming that dTRAF2 and Grnd contribute to apoptosis in egrweak cells.”

    *1.5 Minor The ability of the wing disc to regenerate has been associated with the induction of a developmental delay mediated by Dilp8. Are the authors observing this developmental delay is the case of sal-lexA>rpr + Ap-gal4 >wgn RNAi or sal-lexA>rpr + Ci-Gal4>wgn RNAi *

    The developmental delay due to Dilp8 has been observed by many laboratories, indeed. The question of the reviewer is relevant because if there is no delay in pupariation, regeneration could be compromised not because regeneration has been affected but because after pupariation regeneration is impeded.

    However, delay in pupariation has been found in our experiments. Usually for 11hrs of heat shock (to induce apoptosis) we found 1-2 days of delay.

    We have added the following text:

    “The ability of the wing disc to regenerate after genetic ablation has been associated with the induction of a developmental delay (Colombani et al., 2012; Garelli et al., 2012; Jaszczak et al., 2015; Katsuyama et al., 2015; Smith-Bolton et al., 2009). All genotypes analyzed in figure 6 showed a similar developmental delay of 1-2 days (at 17ºC) after genetic ablation in comparison to the animals of the same genotype in which no genetic ablation was induced, i.e. developed continuously at 17ºC (Fig. S4A). After the adults emerged, the wings were dissected, and regeneration was analyzed.”

    *1.7 Minor - The investigation of the evolutionary origin of TNFR in drosophila included in Fig.2 is cutting a bit the flow of the results. *

    The evolutionary origin starts now with a sentence that can smoothen the flow and few changes in that paragraph have been made:

    “Opposing roles between proteins of the TNFR superfamily suggests that they have an ancient origin and have followed divergent evolutionary paths. To track the differences observed between grnd and wgn, we decided to investigate the evolutionary origin of these two Drosophila genes.”

    *1.8 Minor The authors could explain in more details the double transactivation system for non-fly specialists. *

    The entire section has been re-written in Material and Methods.

    *1.9 Minor - It can be interesting to include and/or discuss these few references: *

    *PLoS Genet. 2019 Aug; 15(8): e1008133. ** PLoS Genet. 2022 Dec 5;18(12):e1010533. FEBS Lett. 2023 Oct;597(19):2416-2432. *

    *Curr Biol. 2016 Mar 7;26(5):575-84. *

    *Nat Commun. 2020 Jul 20;11(1):3631. **

    All these references, and few others, have been introduced in the text.

    __Reviewer #2 __ *2. 1. The authors find that wgn RNAi enhances hh>egr-induced apoptosis. They validate the results with two independent RNAi lines in Figure S1. However, Figure S1 is missing a control without wgn RNAi, and therefore, the results are difficult to assess. *

    Fig S1A now contains this control.

      1. Are the two independent wgn RNAi lines targeting different regions of the coding sequence? *

    As the regions targeted by the 2 RNAi’s are different, see below, we have included in the text:

    “This observation was corroborated with an independent RNAi-wgn strain targeting a different region in the coding sequence (Fig. S1A and S1B). “

    Bloomington BL55275 (dsRNA-HMCO3962)

    VDRC GD9152 (dsRNA-GD3427)

    *2.7. In Figure 4, the authors show that egr expression induces ROS and performs anti-oxidant experiments. This part could be strengthened if they show that the ROS sensor signal disappears after Sod::Cat expression. *

    We had done this experiment and there is a definitively drop in Mitosox in discs in which the weak allele of *egr *is active. We have included this new image in Figure 4G and in the text.

    *2.8. How effective is egr RNAi? In Figure 5E, F, the authors knock down egr and obtain negative results. Based on this, the authors argue that Wgn localization occurs through an egr-independent mechanism. Drawing strong conclusions based on a negative result with egr RNAi is not a good practice since one cannot rule out residual egr activity that mediates the effect (of course , because there is cell death as well, death cells express egr). I suggest either finding ways to completely abolish egr function, or tone down the conclusion. *

    We have used ‘after knocking down eiger’ instead of in the ‘absence’ or ‘abolish’ eiger.

      1. Figure 6 shows that wgn RNAi aggravates the reaper phenotype. What's missing is a control that expresses wgn RNAi but not reaper. *

    Control experiments using the *UAS-wgnRNAi *in the absence of *rpr *are now shown in figure S4C.

    __Reviewer #3 ____ __*3.1.Minor Fig 6C-E would need a control disc without induced apoptosis (ie wildtype disc) stained for phospho-p38 as a baseline comparison. This is important to judge the significance of the remaining phospho-p38 in panel E where wgn is knocked down. The authors write ** **" However, after knocking down wgn, phosphorylated p38 in the wing pouch ** surrounding the apoptotic cells was abolished (Fig. 6E)." **Depending on the amount of phospho-p38 in control discs, this may need to be rephrased to "strongly reduced" instead of "abolished". *

    A control disc stained with P-p38 has been added in Figure 6.

    We have changes ‘abolished’ by ‘strongly reduced’.

    3.2. This sentence in the Intro needs fixing because TNFa doesn't transduce the signal from TNFR to Ask1 since it's upstream of TNFR: "TNFα can transduce the TRAF-mediated signal from TNFR to Ask1 to modulate its activity (Hoeflich et al., 1999; Nishitoh et al., 1998, p. 0; Obsil & Obsilova, 2017; Shiizaki et al., 2013)." *

    We have rephrased this sentence by:

    “TNFα binds to TNFRs which in turn interact with TRAFs to transduce the signal to Ask1 to modulate its activity”.

    *3.3a In the results section, the authors start by ectopically overexpressing Eiger. Are there conditions where Eiger expression is induced in the wing? If yes, it would be helpful for the reader to mention that this system with the genetically GAL4-induced expression of Eiger aims to phenocopy one of these conditions. *

    Eiger ectopic expression has been induced in the wing to generate apoptosis. This is a common technique in Drosophila, and the Reviewer3 is right that a sentence should be useful for the reader.

    A sentence has been introduced at the beginning of the results section:

    “Ectopic expression of *egr *in Drosophila imaginal discs results in JNK-dependent apoptosis (Brodsky et al., 2004; Igaki et al., 2002; Moreno, Yan, et al., 2002).”

    *3.3b Fig 2C is not very self-explanatory: it is worth writing out what Hsa (H. sapiens), Bla and Sco stand for (there is plenty of space). *

    We have re-designed figure 2 to make it more self-explanatory.

    *3.4. This sentence is confusing: ** " ...Wgn localization were due to ROS or to the expression of egr, we used RNAi to knock down egr in the apoptotic cells and found that reduced Egr/TNFα had no effect on Wgn localization (Fig. 5E, 5F)." The authors may want to specify that Wgn is still accumulated even without Egr. ("No effect" is unclear). *

    This sentence has been modifies as:

    “Wgn localization were due to ROS or to the expression of egr, we used RNAi to knock down egr in the apoptotic cells and found that Wgn accumulation was not altered by the knocking down Egr/TNFα (Fig. 5E, 5F). “

    *3.5 Comment. It discovers that Wengen is activated by ROS. In fact, since Wengen binds TNF with an affinity that is several orders of magnitude lower than Grindelwald, and since Wengen is not even located at the cell membrane, these data call into question whether Wengen is a TNF receptor, or a ROS receptor? Could the authors comment on this ? Could it be that the results obtained in the past showing that Wengen is activated by TNF were actually due to TNF inducing apoptosis, leading to production of ROS, leading to activation of Wengen?

    We totally agree with Reviewer#3. We have added a final paragraph in the discussion section.

    “We speculate that the subcellular location of Wgn and Grnd may contribute to the different functions of both receptors. Grnd is more exposed at the apical side of the plasma membrane, which makes this receptor more accessible for ligand interactions (Palmerini et al., 2021). Wgn, embedded in cytoplasmic vesicles, is less accessible to the ligand and could be more restricted to being activated by local sources of signaling molecules, such as ROS. In contrast to initial reports (Kanda et al., 2002; Kauppila et al., 2003), los-of-function of wgn does not rescue Egr-induced apoptosis in the Drosophila eye (Andersen et al., 2015), which supports our observation in the wing that Wgn is not required for Egr-induced apoptosis. Instead, Egr-induced apoptosis generates ROS which target intracellular Wgn to foster a cell survival program of cells close to the apoptotic zone.”

    __Reviewer #4 __

    *4.1 b Are phospho-p38 levels increased in cells expressing Egr[weak]? *

    We have the results of these experiments. To respond to this point, a new figure has been added (Fig. S4) in which we show the P-p38 levels are increased (non-autonomously) in egrw, as previously found for reaper. In addition, we show that egrw + activation of p35 and egrw + activation of Sod1::Cat results in strong reduction of P-p38. This indicates that P-p38 is stimulated by the ROS produced by apoptotic cells.

    The text now:

    “It is worth noting that cells egrw induce phosphorylation of p38 in neighboring cells (Fig. S4A) and that, as previously found for rpr (REF), depends on ROS generated by egrw apoptotic cells (Fig. S4B, C).”

    *4.2 In Figure 4C it appears that the Dcp-1 positive cells move apically rather than basally. Including nuclear staining would be very informative allowing assessment of tissue morphology. ** The authors focus on the pouch region of the wing imaginal disc, where phenotypes are strong and obvious. However, the hh-Gal4 driver also affects posterior cells in the hinge and notum, where the effects of Eiger[weak] overexpression seem weaker (e.g., minimal to no MitoSox signal in hinge and notum posterior cells). Could the authors explain this observation? *

    Point 1: Actually, cells move more basally, though some move more apical as well. Depending on the section cells the image could be confusing. To solve that, we show now a plane on these discs at apical and a plane basal. Both high magnifications. There one can see that there is more concentration of pyknotic nuclei basally. We have added this observation in a new supplementary figure (Fig. S3) and the corresponding text in page 5: “Apoptotic cells in egrweak are characterized by pyknotic nuclei and are positive for Dcp1. These cells tend to concentrate in the basal side of the epithelium, although some are scattered apically (Fig. S3). Accumulation of Wgn was observed in healthy anterior cells adjacent to both apical and basal egrweak cells (Fig. 4, Fig. S3A, B).”

    Point 2 Comment on MitoSOX in notum: At the stages of the imaginal discs used in this study, almost all notum cells are anterior compartment. The hh positive cells in notum much less abundant, therefore most of the staining was found in the posterior compartment of the wing pouch.

    *4.5 Figure 6 C-E. Does WgnRNAi potentiates and GrndRNAi suppress Rpr-induced apoptosis similarly to their effects when knocked down in Eiger[weak]OE cells? *

    The areas controlled by salE/Pv >rpr (dotted lines) are full of pycnotic nuclei, which indicates concentration of apoptotic cells in all genotypes shown.

    Thus, in the conditions generated here, apoptosis is not inhibited and grnd RNAi does not interfere with the activation of P-p38. In wgn knock down, phospho-p38 is strongly inhibited, indicating the importance of wgn in phosphorylation of p38.

    To clarify this point, we have added in the text: “Note that rpr-induced apoptosis is not suppressed after knocking down grnd or wgn.” Also in the figure legend we added: “White lines in the confocal images outline the salE/Pv-LHG,LexO-rpr dark area full of pyknotic nuclei of apoptotic cells.”

    4.6 The activation of p38 following salE/Pv>rpr-mediated ablation as shown by immunostaining is noteworthy. While loss Grnd knockdown leads to phospho-p38 signal enrichment around the rpr-expressing cells, WgnRNAi results in reduced phospho-p38 signal in the wing pouch but also beyond the nub-expression domain. Do salE/Pv>rpr nub>WgnRNAi cells still generate ROS?

    So far there is no evidence of Wengen as a ROS scavenger. We have evidence that ROS (using MitoSox probe) are produced in egrweak + Wgn RNAi cells. Thus, the inhibition of wgn expression does not block ROS production. A new figure shows this observation (Figure S4A).

    4.7 Are ROS responsible for the long-range signaling and p38 activation, referring to authors' previous work Santabarbara-Ruiz et al., 2019, PLoS Genet 15(1): e1007926. https://doi.org/10.1371/journal. pgen.1007926, Figure 5G?

    ROS are responsible for p38 activation as shown in a new figure (Fig. S4). In this new figure egrweak is activated in hh, and p38 is most of cells in the posterior compartment, and also anterior. However, after blocking apoptosis or ROS production, this P-p38 is reduced.

    4.8 Minor I propose rephrasing the description of "UAS-Egr[weak] transgene, a strain that produces a reduced Egr/TNFα activity". It could imply a loss of function strain rather than a transgene that causes mild/moderate Egr overexpression.

    The sentence has been rephrases as suggested (End of the first paragragraph in results section).

    *4.9 Minor. I recommend the authors to revise the charts for improved clarity in genotype representation. For example, in Figure 1I, the label "control-GFP" might be misleading. It would be beneficial to specify that "control" refers to Eiger[weak] alone with other manipulations being done simultaneously with Eiger[weak] overexpression. *

    All charts have been revised.

    *4.10 Minor. Additionally, considering that individuals with color blindness may struggle to differentiate between red and green colors, I strongly suggest using a color-blind-friendly palette, especially in Figure 4A, C, G, and Figure 4A, C, E." ** *All images have been revised for color blind code.

    • 11 Minor. Providing detailed information regarding the reagents used in the study, such as Catalogue Numbers or RRIDs, is beneficial for enhancing reproducibility. *

    We have added the RRID and Cat #. If no ID was available, we added the reference or contact.

    4.12 This reviewer points two limitations that we are now trying to solve:

    *Limitations: *

    *Quality of the imaging – higher magnification images and quantification would enhance the study. ** The summarizing model may contain excessive speculations that lack support from the data or references to the existing literature. *

    Quality of imaging. We have now an extra supplemental figure with higher magnifications. Extra higher magnifications will be included in the next version as well as quantification, as exposed for the Revision Plan points 4.3 and 4.4.

    Model: We have re-written the paragraph on the model, introduced references and drop some speculations. We hope the current version will be more convincing for the reader.

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

    Please include a point-by-point response explaining why some of the requested data or additional analyses might not be necessary or cannot be provided within the scope of a revision. This can be due to time or resource limitations or in case of disagreement about the necessity of such additional data given the scope of the study. Please leave empty if not applicable.

    Reviewer 1

    *1.3. Is the overexpression of Wengen sufficient to increase tissue regeneration? *

    The suggestion of the reviewer is a key point in regeneration biology: how to accelerate regeneration?

    We have demonstrated that Wengen is upstream the Ask1-p38 axis that drives regeneration. The reviewer wonders if Wengen overexpression can result in increase in regeneration. In a previous work we have demonstrated that p38 activation is key for regeneration but its overexpression can be deleterious (Esteban-Collado et al., 2021). Only in discs that sensitized for low p38 (starvation, low Akt, Ask1S83A mutant), the overexpression rescues regeneration. Therefore, the levels of the Wgn-Ask1-p38 have to be very tightly controlled. An excess will be deleterious. We are aware of the importance of the question, but at this point we do not have the technology to finely control Wgn-Ask1-p38 levels to do this experiment.

    1.6 Minor - It possible to test the induction of apoptosis in a wgn null mutant background to see if the phenotype is even stronger than the one observed with RNAi (the wgn RNAi is induced at the same time than egr or rpr overexpression).

    Flies wgnKO survive, but they gave us problems when carrying transgenes for our design of genetic ablation. Indeed, we tried to generate wgnKO carrying Gal4+tubGal80+eigerweak without success.

    In addition, the reason we have used an inducible mutant is because it allows us to work in time and space without altering expression in other cell types beyond wing discs. Wgn is required in other organs during development like gut, trachea and axon growth, etc.., and thus, we ensure the affected cells belong to the tissue analyzed.

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    Referee #4

    Evidence, reproducibility and clarity

    The study by Florenci Serras and colleagues presents compelling evidence highlighting distinct functions of the two Drosophila TNFRs, Wengen (Wgn) and Grindewald (Grnd) in developing imaginal epithelia. The study shows that while Grnd-dTraf2-Tak1 module controls apoptosis in Eiger (Egr)-dependent manner, Wgn-dTraf1-Ask1 promotes survival likely via p38 signaling independent of Egr. Their phylogenetic analysis underscores the ancient origin of both receptors while revealing their divergent evolutionary path, manifested by markedly different CRD sequences. Moreover, Wgn shows higher degree of similarity to mammalian TNFRs. Using functional genetics and confocal imaging of immunostained wing imaginal discs, the authors confirm the differential localization of Wgn and Grnd in imaginal cells, consistent with several recent studies. Interestingly, they demonstrate distinct responses between Grnd that is internalized upon Eiger[weak] overexpression from the plasma membrane, and Wgn, which cytoplasmic levels decrease in Eiger[weak] OE cells but become enriched in neighboring wild type cells. The non-autonomous accumulation of Wgn required ROS but not Eiger production by dying cells. Finally, employing an elegant double-driver lexA/lexO and Gal4/UAS system, enabling independent gene manipulation in specific domains of the wing imaginal discs, the authors established the essential role of Wgn, but not Grnd, in the regenerative response to apoptosis, including the occurrence of phosphorylated p38.

    Major comments:

    The conclusion regarding the protective role of p38 in response to Egr[weak] should be supported by a p38 knockdown experiment. Are phospho-p38 levels increased in cells expressing Egr[weak]?

    In Figure 4C it appears that the Dcp-1 positive cells move apically rather than basally. Including nuclear staining would be very informative allowing assessment of tissue morphology. The authors focus on the pouch region of the wing imaginal disc, where phenotypes are strong and obvious. However, the hh-Gal4 driver also affects posterior cells in the hinge and notum, where the effects of Eiger[weak] overexpression seem weaker (e.g., minimal to no MitoSox signal in hinge and notum posterior cells). Could the authors explain this observation?

    In Figure 5, the cells expressing Rpr appeared to be pulled/extruded basally as expected. It would be beneficial to quantify Wgn and Grnd signals along cross-sections and provide higher magnification images of domain boundaries to illustrate differences in TNFR localization and levels. The micrographs for Grnd Figure 5B,D, F capture substantial signal from the peripodial epithelium where the salE/Pv> driver is likely not active?

    The non-autonomous induction of Wgn seems stronger when facing dying Rpr overexpressing cells simultaneously depleted of Eiger compared to RprOE alone. Should this be a reproducible, could the authors discuss potential reason for this observation?

    Figure 6 C-E. Does WgnRNAi potentiates and GrndRNAi suppress Rpr-induced apoptosis similarly to their effects when knocked down in Eiger[weak]OE cells? The activation of p38 following salE/Pv>rpr-mediated ablation as shown by immunostaining is noteworthy. While loss Grnd knockdown leads to phospho-p38 signal enrichment around the rpr-expressing cells, WgnRNAi results in reduced phospho-p38 signal in the wing pouch but also beyond the nub-expression domain. Do salE/Pv>rpr nub>WgnRNAi cells still generate ROS? Are ROS responsible for the long-range signaling and p38 activation, referring to authors' previous work Santaba ́rbara-Ruiz et al., 2019, PLoS Genet 15(1): e1007926. https://doi.org/10.1371/journal. pgen.1007926, Figure 5G?

    Minor comments:

    I propose rephrasing the description of "UAS-Egr[weak] transgene, a strain that produces a reduced Egr/TNFα activity". It could imply a loss of function strain rather than a transgene that causes mild/moderate Egr overexpression.

    I recommend the authors to revise the charts for improved clarity in genotype representation. For example, in Figure 1I, the label "control-GFP" might be misleading. It would be beneficial to specify that "control" refers to Eiger[weak] alone with other manipulations being done simultaneously with Eiger[weak] overexpression. Additionally, considering that individuals with color blindness may struggle to differentiate between red and green colors, I strongly suggest using a color-blind-friendly palette, especially in Figure 4A, C, G, and Figure 4A, C, E."

    Providing detailed information regarding the reagents used in the study, such as Catalogue Numbers or RRIDs, is beneficial for enhancing reproducibility.

    Significance

    This is a very solid study that uncovers unique roles of Drosophila TNFRs in regulating imaginal cell behaviors crucial for tissue regeneration. It expands our knowledge on processes controlled by TNFR-mediated signaling, highlighting the potential for ligand-independent regulation. The study nicely complements recent findings by several laboratories (Letizia et al., 2023; Loudhaief et al., 2023; Palmerini et al., 2021). Beyond its contribution to fundamental biology, the study has biomedical implication for regenerative medicine. It emphasizes the necessity of balancing TNFR activities, downstream signaling and their dependence on ligands, providing important insights for the development of receptor agonists or antagonists. The findings are relevant to audience interested in developmental and regenerative biology, gene evolution.

    Strengths: functional genetics revealing distinctive roles for the two TNFRs in Drosophila and their dependency on ligand in the paradigm of tissue regeneration.

    Limitations: quality of the imaging - higher magnification images and quantification would enhance the study. The summarizing model may contain excessive speculations that lack support from the data or references to the existing literature.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    TNF receptors have a broad range of possible function, from inducing apoptosis to promoting cell survival and proliferation. How this works is not completely understood. Drosophila has two TNF receptors, Wengen and Grindelwald. This manuscript nicely shows that Grindelwald is pro-apoptotic while Wengen promotes cell survival and proliferation. Strikingly, if TNF is expressed in Drosophila tissue, knockdown of the receptor Wengen leads to elevated levels of apoptosis, clearly showing its cell-protective function. Interestingly, the authors find that Wengen is activated by ROS produced by neighboring dying cells - regardless of whether they are dying due to TNF signaling or not - and that Wengen then activates p38 downstream to mediate a regenerative response.

    Major comments:

    Overall the conclusions are interesting, clear and convincing. The data are of very good quality. I only have a few minor comments below.

    Minor comments:

    1. Fig 6C-E would need a control disc without induced apoptosis (ie wildtype disc) stained for phospho-p38 as a baseline comparison. This is important to judge the significance of the remaining phospho-p38 in panel E where wgn is knocked down. The authors write " However, after knocking down wgn, phosphorylated p38 in the wing pouch surrounding the apoptotic cells was abolished (Fig. 6E)." Depending on the amount of phospho-p38 in control discs, this may need to be rephrased to "strongly reduced" instead of "abolished".
    2. This sentence in the Intro needs fixing because TNFa doesn't transduce the signal from TNFR to Ask1 since it's upstream of TNFR: "TNFα can transduce the TRAF-mediated signal from TNFR to Ask1 to modulate its activity (Hoeflich et al., 1999; Nishitoh et al., 1998, p. 0; Obsil & Obsilova, 2017; Shiizaki et al., 2013)."
    3. In the results section, the authors start by ectopically overexpressing Eiger. Are there conditions where Eiger expression is induced in the wing? If yes, it would be helpful for the reader to mention that this system with the genetically GAL4-induced expression of Eiger aims to phenocopy one of these conditions.
    4. Fig 2C is not very self-explanatory: it is worth writing out what Hsa (H. sapiens), Bla and Sco stand for (there is plenty of space).
    5. This sentence is confusing: " ...Wgn localization were due to ROS or to the expression of egr, we used RNAi to knock down egr in the apoptotic cells and found that reduced Egr/TNFα had no effect on Wgn localization (Fig. 5E, 5F)." The authors may want to specify that Wgn is still accumulated even without Egr. ("No effect" is unclear).

    Significance

    This manuscript makes several important discoveries:

    1. it clearly shows that one TNF receptor, Grindelwald, is mainly pro-apoptotic, while the other, Wengen, is mainly pro-survival. This provides a mechanistic explanation for the dual role of the TNF, Eiger.
    2. It discovers that Wengen is activated by ROS. In fact, since Wengen binds TNF with an affinity that is several orders of magnitude lower than Grindelwald, and since Wengen is not even located at the cell membrane, these data call into question whether Wengen is a TNF receptor, or a ROS receptor? Could the authors comment on this ? Could it be that the results obtained in the past showing that Wengen is activated by TNF were actually due to TNF inducing apoptosis, leading to production of ROS, leading to activation of Wengen?
    3. It was previously shown that damage, for instance in the fly intestine, induces production of ROS, which then activates p38, leading to a proliferative/regenerative response. This manuscript provides a missing mechanistic link, showing that the ROS activates Wengen, which in turn activates p38. This thereby completes the mechanistic chain of events from damage to the regenerative response.

    Hence, overall, this is a very interesting study. It will be of interest for a broad audience of people studying TNF signaling, stress signaling and stress response, tissue damage and repair, and regeneration.

    My expertise: Drosophila, growth, signaling

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    Referee #2

    Evidence, reproducibility and clarity

    The Drosophila genome encodes a single TNFa ortholog, eiger (egr), and two TNF Receptros (TNFR), wengen (wgn) and grindelwald (grnd). While egr overexpression can cause apoptosis, the authors here report that wgn and grnd have opposing roles in cell death and survival. Specifically, the authors show evidence that grnd promotes cell death in response to egr expression, while wgn promotes cell survival through the p38 MAP Kinase pathway. They further show that apoptotic cells have high levels of ROS, which activates the wgn-p38 axis for tissue regeneration, independent of egr.

    Overall, the manuscript is well written. At the same time, there are some technical concerns and missing controls that need to be addressed. Below are a few specific comments for the authors' consideration:

    Major Comments

    1. The authors find that wgn RNAi enhances hh>egr-induced apoptosis. They validate the results with two independent RNAi lines in Figure S1. However, Figure S1 is missing a control without wgn RNAi, and therefore, the results are difficult to assess.
    2. Are the two independent wgn RNAi lines targeting different regions of the coding sequence?
    3. Aside from wgn, other RNAi experiments are not validated through independent RNAi lines. I suggest expanding the Supplemental Figures to reproduce a few key findings with independent RNAi lines.
    4. In Figure 1E, the authors show that wgn RNAi enhances cell death caused by hh>egr. What is missing here is a wgn RNAi control without hh>egr. Is there any cell death caused by the loss of wgn alone (without hh>egr)?
    5. If wgn RNAi causes some degree of cell death, is the observed effect with hh>egr a significant genetic interaction, or merely additive?
    6. Is the wgn-p38 pathway sufficient to block egr induced cell death? The authors could test this by having hh>egr in the licT1.1 background. The authors have a more complex experiment in Figure 3, where licT1.1 is introduced into the hh>egr, wgn RNAi background. However, testing the effect of licT1.1 without wgn would establish a more direct relationship between egr and wgn-p38.
    7. In Figure 4, the authors show that egr expression induces ROS and performs anti-oxidant experiments. This part could be strengthened if they show that the ROS sensor signal disappears after Sod::Cat expression.
    8. How effective is egr RNAi? In Figure 5E, F, the authors knock down egr and obtain negative results. Based on this, the authors argue that Wgn localization occurs through an egr-independent mechanism. Drawing strong conclusions based on a negative result with egr RNAi is not a good practice since one cannot rule out residual egr activity that mediates the effect. I suggest either finding ways to completely abolish egr function, or tone down the conclusion.
    9. Figure 6 shows that wgn RNAi aggravates the reaper phenotype. What's missing is a control that expresses wgn RNAi but not reaper.

    Significance

    There is now a detailed understanding of mammalian TNFRs, which play pro-apoptotic and non-apoptotic roles depending upon the context. Previous studies had also reported that TNFR1 respond to ROS. By comparison, our understandings of the two TNFRs in Drosophila remain rudimentary. The two receptors have different loss-of-function phenotypes, some of which may be independent of egr signaling. The major significance of this work is in delineating the distinct behaviors of the two Drosophila TNFRs, centering around their pro-apoptotic, or pro-survival properties.

    Audience: This study will draw the interest of Drosophila geneticists, those interested in Reactive Oxygen Species and cell death, and evolutionary biologists.

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    Referee #1

    Evidence, reproducibility and clarity

    Tumor necrosis factor (TNF)-α stands out as a remarkably conserved pro-inflammatory cytokine that plays crucial roles in immunity, tissue repair, and cellular homeostasis. The Drosophila TNF-TNF receptor (TNFR) system, known for its simplicity, combined with a versatile genetic toolkit, has been instrumental in unraveling the intricate mechanisms governing both the physiological and pathological functions mediated by TNF. Recently, the fly TNFR Wengen has been described to have ligand independent functions in maintaining tissue homeostasis and tracheal remodeling. The current manuscript describes a novel TNF/Egr-independent function of Wengen in regulating tissue regeneration in imaginal discs. The authors identify both the upstream regulator (ROS) and the downstream signaling pathway through Ask1/p38 MAPK. The data presented are solid and support an interesting model where ROS emanating from damaged tissue triggers Wgn-dependent signaling in adjacent cells to promote regeneration. Few points could be addressed:

    Major points:

    • The result in Fig1.H is somehow surprising. How does the overexpression of Egr induce caspase activation in the absence of its receptor Grnd? Is it possible that the loss of function of Wengen on its own has a phenotype? If so, that would suggest that Wgn in addition to its role in regeneration might be implicated in pro-survival processes in homeostatic conditions?
    • In Fig.6, it would be relevant to include wengen inactivation within the domain where rpr is expressed to show that wengen is not required autonomously for regeneration (sal>rpr + wgn RNAi). What is the phenotype of the adult wing of sal-lexA>rpr + nub-gal4 >wgn RNAi animals?
    • Is the overexpression of Wengen sufficient to increase tissue regeneration?

    Minor points:

    • In fig.1I, it is surprising that knockdown of neither Grnd nor dTRAF2 significantly affects Egr-induced apoptosis
    • The ability of the wing disc to regenerate has been associated with the induction of a developmental delay mediated by Dilp8. Are the authors observing this developmental delay is the case of sal-lexA>rpr + Ap-gal4 >wgn RNAi or sal-lexA>rpr + Ci-Gal4>wgn RNAi
    • It possible to test the induction of apoptosis in a wgn null mutant background to see if the phenotype is even stronger than the one observed with RNAi (the wgn RNai is induced at the same time than egr or rpr overexpression).
    • The investigation of the evolutionary origin of TNFR in drosophila included in Fig.2 is cutting a bit the flow of the results.
    • The authors could explain in more details the double transactivation system for non-fly specialists.
    • It can be interesting to include and/or discuss these few references:

    PLoS Genet. 2019 Aug; 15(8): e1008133.

    PLoS Genet. 2022 Dec 5;18(12):e1010533.

    FEBS Lett. 2023 Oct;597(19):2416-2432.

    Nat Commun. 2020 Jul 20;11(1):3631.

    Curr Biol. 2016 Mar 7;26(5):575-84.

    Significance

    The understanding of the mechanistic interplay between TNFR in integrating TNF-dependent and independent signals to stimulate distinct downstream responses lays the foundation for investigating whether these insights can be generalized to other members within the TNFR superfamily in all organisms. This work is relevant for a large audience of researchers working in the field of inflammation and TNFR.

  6. Version published to 10.1101/2023.11.13.566843v1 on bioRxiv