Adhesion to a common ECM mediates interdependence in tissue morphogenesis in Drosophila

  1. LE Sánchez-Cisneros
  2. M Barrera-Velázquez
  3. Dimitri Kromm
  4. P Bun
  5. H Merchant-Larios
  6. LD Ríos-Barrera

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Abstract

For organs to be functional, the cells and tissues that constitute them must effectively interact with each other and coordinate their behaviours. Halfway during Drosophila embryogenesis, two lateral epidermal sheets stretch to fuse at the dorsal midline; concomitant with this, the main tubes of the respiratory system also shift dorsally. Here, we show that these processes occur simultaneously and are coordinated by the adhesion of the epidermal sheets and a subset of cells of the tracheal trunks to a common extracellular matrix (ECM) that separates them. We show that during dorsal closure, tracheal trunk cells extend protrusions towards the ECM underneath the epidermis. These protrusions are under tension, suggesting they have a mechanical function. Additionally, perturbing adhesion between tracheal cells and the epidermis affects the development of both tissues. Altogether, our findings uncover a novel mechanism used for tissue coordination during development, one that is based on tissue adhesion towards a common ECM capable of transmitting forces across the embryo.

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    Reply to the reviewers

    Reply to the reviewers

    We would like to thank the reviewers for their comments, we see great value in the suggestions they made to strengthen our work. We are glad to see that they are in general positive about the manuscript. In the following, we include a point-by-point response to their comments, which are in general consistent with each other.

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    In this manuscript, Sanchez-Cisneros and colleagues, examine how tracheal cell adhesion to the ECM underneath the epidermis helps shape the tracheal system. They show that if cell-ECM adhesion is perturbed the development of the tracheal system and the epidermis is disrupted. They also detect protrusions extending from the dorsal trunk cells towards the ECM. The work is novel, the figures are clear, and the questions are well addressed. However, I find that some of the claims are not completely supported by the data presented. I have some suggestions that will, I believe, clarify certain points.

    Major comments

    At the beginning of the results section as in the introduction the authors claim that "It is generally assumed that trunk displacement occurs due to tip cells pulling on the trunks so that they follow their path dorsally." This sentence is not referenced, and I do not know where it has been shown or proposed to be like this. In addition, the comparison with the ventral branches is also not referenced and the movie does not really show this. Forces generated by tracheal branch migration have been shown to drive intercalation (Caussinus E, Colombelli J, Affolter M. Tip-cell migration controls stalk-cell intercalation during Drosophila tracheal tube elongation. Curr Biol. 2008;18(22):1727-1734. doi:10.1016/j.cub.2008.10.062), but not dorsal trunk (DT) displacement.

    We agree that dorsal trunk displacement has not been discussed in previous works, just the fact that tip-cell migration influences stalk cell intercalation. We will rephrase this sentence, stating that dorsal trunk displacement has not been studied.

    However, to rule out the possibility that DT displacement and the phenotype observed in XXX is due to dorsal branch pulling forces, the authors should analyze what happens in the absence of dorsal branches (in condition of Dpp signalling inhibition as in punt mutants or Dad overexpression conditions).

    This is a great idea, and we thank the reviewer for suggesting this. We tried to achieve a similar goal by expressing a Dominant Negative FGFR (Breathless-DN) in the tracheal system, since its expression under btl-gal4 affects tip cell migration. However, the phenotype arises too late to have an effect in dorsal branch migration during the stages we were interested in analyzing. The alternative proposed by the reviewer should be more efficient, as blocking Dpp signalling prevents the formation of dorsal branches completely. We have just received flies carrying the UAS-Dad construct. We will express Dad under btl-gal4 and see how this affects dorsal trunk displacement.

    I am concerned about the TEM observations. The authors claim they can identify tracheal cells by their lumen (Fig. 2 C'). However, at stage 15, the tracheal lumen should be clearly identifiable, and the interluminal DT space should be wider relative to the size of the cells. In this case, there is nothing telling us that we are not looking at a dorsal branch or lateral trunk cell. Furthermore, at embryonic stage 15, the tracheal lumen is filled with a chitin filament, which is not visible in these micrographs. Also, there is quite a lot of tissue detachment and empty spaces between cells, which might be a sign of problems in sample fixing. Better images and more accurate identification of dorsal trunk cells is necessary to support the claim that "These experiments revealed a novel anatomical contact between the epidermis and tracheal trunks".

    The protocol that we use for TEM involves performing 1-μm sections that allow us to stage embryos and to identify the anatomical regions using light microscopy and then switch to ultra-thin sections for electron microscopy once we have found the right position within the sample. This approach also allows us to determine the integrity of the sample. We attach here a micrograph of the last section we analyzed before we decided to do the EM analysis. The asterisk (*) points to a region where the multicellular lumen of the trunk is visible. Due to its proximity to the posterior spiracles, we are confident this is the dorsal trunk and not the lateral trunk. We realize now, after comparing this image with an atlas of development (Campos-Ortega and Hartenstein, 2013), that the stage we chose to illustrate the interaction is a stage 14 embryo instead of the stage 15 we indicated in the manuscript. We will change the stage but given that dorsal closure has already started by stage 14, this does not affect our analysis. Still, we apologize for the mis-staging of the embryo.

    In the light-microscopy image, we have overlaid the EM section to the corresponding region of interest. We agree that the lumen should be thicker compared to the length of the cells, if the section would be cutting the trunk through its largest diameter. However, the protrusions we see do not emerge from the middle part of the trunk where the lumen is found but are seen towards the dorsal side of the trunk, where the lumen will no longer be visible in a longitudinal section as the ones we present. In the embryo shown in Figure 2A-C, our interpretation is that the section was done through a very shallow section of the lumen (represented below). We interpret this from the fact that we see abundant electron-dense areas which we think are adherens junctions from multiple cells. These junctions are visible in Figure 2C but are currently not labelled. We will add arrows to increase their visibility.

    Given that protruding cells lie at the base of dorsal branches, it would be expected that in some sections we would find the protrusions close to the dorsal branches. This is in fact what we show in the micrograph shown in Figure 2D, with a lower magnification overview image shown in Figure S2D. In this case, we see a cell in close proximity to the tendon cells on one side (Figure 2D), which is connected to a dorsal branch on the opposite side (shown in Figure S2D). This dorsal branch is clearly autocellular and chitin deposition is visible as expected for the developmental stage. Again, in Figure S2E we see an electron-dense patch near the lumen that corresponds to the adherens junctions that seal the lumen. We see that all this needs to be better explained in the manuscript, so we will elaborate on the descriptions, and incorporate the light microscopy micrograph to the supplemental figures. This should also aid with the anatomical descriptions requested by Reviewer #3. Nevertheless, we think these observations confirm that what we are describing are the contact points between the dorsal trunk and tendon cells.

    Timelapse imaging of the protrusions in DT cells is done with frames every 4 minutes (Video S3). This is not enough to properly show cellular protrusions and the images do not really show interaction with the epidermis. Video S4 has a better time resolution but it is very short and only shows the cut moment. Video S4, shows the cut, but the reported (and quantified recoil) is not clear. Nevertheless, the results are noteworthy and should be further analysed.

    We will acquire high temporal resolution time-lapse images using E-Cadherin::GFP and btl-gal4, UAS-PH::mCherry to show the behaviour of the protrusions on a short time scale.

    Provided these embryos survive, would it be possible to check if embryos after laser cutting will develop wavy DTs?

    We think it would be interesting to carry out this experiment, but the laser cut experiments were done under a collaborative visit and we would not be able to repeat it in a short-term period.

    What happens to the larvae under the genetic conditions presented in Fig.S3? Do they reach pupal stages? Do these animals reach adult stages?

    We have seen escapers out of these crosses, but we have not quantified the lethality of the experiment. We will analyse this and include it in the manuscript.

    The kayak phenotypes are very interesting and perhaps the authors could explore them more. As in inhibition of adhesion to the ECM, kay mutants display wavy dorsal trunks. Do they have defective adhesion? Fos being a transcription factor, this is a possibility. The authors should at least discuss the kay phenotypes more extensively and present a suitable hypothesis for the phenotype.

    We agree that the kayak experiments might bring more consequences than just preventing dorsal closure. We will complement this approach by blocking dorsal closure by other independent means. We will use pannier-gal4 (a lateral epidermis driver), engrailed-gal4 (a driver for epidermal posterior compartment), and 332-gal4 (an amnioserosa driver) to express dominant-negative Moesin. In our experience, this also delays dorsal closure and it should result in a similar tracheal phenotype as the one we see in kayak embryos.

    Minor comments

    Page 2 Line 9/10 The sentence "tracheal tubes branch and migrate over neighbouring tissues of different biochemical and mechanical properties to ventilate them." should be rewritten. Tracheal cells do not migrate over other tissues to ventilate them.

    We meant to say that tracheal cells migrate over other tissues at the same time as they branch and interconnect to allow gas exchange in their surroundings after tracheal morphogenesis is completed. Ventilation is used here as a synonym for gas exchange or breathing. We will rephrase this if the reviewer considers it confusing.

    Page 2 Line 24/25 The sentence "It has been generally assumed that trunks reach the dorsal side of the embryo because of the pulling forces of dorsal branch migration." needs to be backed up by a reference.

    As explained above, we will rephrase this sentence.

    Page 7 Line 32/23 In this sentence, the references are not related to dorsal closure "Similarly, the signals that regulate epidermal dorsal closure do not participate in tracheal development, or vice versa (Letizia et al., 2023; Reichman-Fried et al., 1994)."

    Our goal in this sentence was to explain that while JNK is required for proper epidermal dorsal closure, loss of JNK signaling in the trachea does not affect tracheal development (as shown by Letizia et al., 2023). At the same time, Reichman-Fried et al., 1994 described the phenotypes of loss of breathless (btl). We will remove this last reference as the work does not study the epidermis. We will rephrase the sentence as: “Similarly, the signals that regulate epidermal dorsal closure do not participate in tracheal development; namely, JNK signaling (Letizia et al., 2023).”

    Page 12 Line 1 "Muscles attach to epidermal tendon cells through a dense meshwork of ECM" this sentence must be referenced.

    We will add the corresponding references for this statement: (Fogerty et al., 1994; Prokop et al., 1998; Urbano et al., 2009). We will change “dense” for “specialized”.

    Fig. S1- Single channel images (A'-C' and A'-C') should be presented in grayscale.

    Fig. S4- Single channel images (A'-D' and A'-D') should be presented in grayscale.

    We will add the grayscale, single-channel images for these figures.

    Reviewer #1 (Significance (Required)):

    The findings shown in this manuscript shed light on the interactions and cooperation between two organs, the tracheal system and the epidermis. These interactions are mediated by cell-ECM contacts which are important for the correct morphogenesis of both systems. The strengths of the work lie on its novelty and live analysis of these interactions. However, its weaknesses are related to some claims not completely backed by the data, some technical issues regarding imaging and some over-interpreted conclusions.

    This basic research work will be of interest to a broad cell and developmental biology community as they provide a functional advance on the importance of cell-ECM interactions for the morphogenesis of a tubular organ. It is of specific interest to the specialized field of tubulogenesis and tracheal morphogenesis.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    Summary: In this paper, the authors explore the relationships between two Drosophila tissues - the epidermis and tracheal dorsal trunk (DT) - that get dorsally displaced during mid-late embryogenesis. The show a nice temporal correlation between the movements of the epithelia during dorsal closure and DT displacement. They also show a correlation between the movement of an endogenously tagged version of collagen and the DT, suggesting that the ECM may contribute to this coordinated movement. Through high magnification TEM, they show that tracheal cells make direct contact with the subset of epithelial cells, known as tendon cells, that also serve as muscle attachment sites. In between these contact sites, tracheae are separated from the epithelia by the muscles. Furthermore, the TEMs and confocal imaging of tracheal cells expressing a membrane marker at these contact sites show that the tracheal cells are extending filopodia toward the tendon cells. The authors then explore how a variety of perturbations to the ECM produced by the tendon and DT cells affect DT and epithelial movement. They find that expressing membrane-associated matrix metalloproteases (MMP1 or MMP2) in tendon cells as well as perturbations in integrin or integrin signaling components leads to delays in dorsal displacement as well as defective lengthening of the tracheal DT tubes. They find that defects in the association between the tracheal and epidermal ECM attachments affect dorsal displacement of the epidermis, disrupting dorsal closure.

    Major comments: I like the goals of this paper testing the idea that the ECM plays important roles in the coordination of tissue placement, and I think they have good evidence of that from this study. However, I disagree with the conclusions of the authors that disrupting contact between DT and the tendon cells has no effect on DT dorsal displacement. DT tracheal positioning is clearly delayed; the fact that it takes a lot longer indicates that the ECM does affect the process. It's just that there are likely backup systems in place - clearly not as good since the tracheal tubes end up being the wrong length.

    We agree with this view; in our deGradFP experiments we see a delayed DT displacement. We focused our analyses on the coordination with epidermal remodelling, which remained unaltered, but we in fact see a delayed progression in dorsal displacement of both tissues (Figure 5I-J). We will emphasize this in the corresponding section of the Results.

    It also seems important that the parts of the DT where the dorsal branches (DB) emanate are moving dorsally ahead of the intervening portions of the trachea. This suggests to me that the DB normally does contribute to DT dorsal displacement and that this activity may be what helps the DT eventually get into its final position. The authors should test whether the portions of the DT that contact the DB are under tension. If the DB migration is providing some dorsal pulling force on the DT, this may also contribute to the observed increases in DT length observed with the perturbations of the ECM between the tendon cells and the trachea - if tube lengthening is a consequence of the pulling forces that would be created by parts of the trachea moving dorsally ahead of the other parts. Here again, it would be good to test if the DT itself is under additional tension when the ECM is disrupted.

    We thank the reviewer for the suggested experiments. We agree with the fact that the dorsal branches should pull on the dorsal trunk and that this interaction should generate tension. Unfortunately, we are unable to test this with the experiments proposed by the reviewer, but we propose an alternative strategy to overcome this. We understand that the reviewer suggests we do laser cut experiments in dorsal branches to see if there is a recoil in the opposite direction of dorsal branch migration. We carried out our laser cut experiments using a 2-photon laser through a visit to the EMBL imaging facility, using funds from a collaborative grant. Funding a second visit would require us to apply for extra funding, which would delay the preparation of the experiments. We are aware of UV-laser setups within our university, however, UV-laser cuts would also affect the epidermis above the dorsal branches, which we think might contribute to recoil we would expect to see.

    Instead of doing laser cuts, we have designed an experiment based on the suggestion of reviewer #1 of blocking Dpp signaling (with UAS-Dad), which would prevent the formation of dorsal branches. We expect that in this experimental setup, the trunk will bend ventrally in response to thepulling forces of the ventral branches. We will also co-express UAS-Dad (to prevent dorsal branch formation) and UAS-Mmp2 (to ‘detach’ the dorsal trunk from the epidermis), and we would expect to at least partially rescue the wavy trunk phenotype.

    Minor comments: The authors need to do a much better job in the intro and in the discussion of citing the work of the people who made many of the original findings that are relevant to this study. Many citations are missing (especially in the introduction) or the authors cite their own review (which most people will not have read) for almost everything (especially in the discussion). This fails to give credit to decades of work by many other groups and makes it necessary for someone who would want to see the original work to first consult the review before they can find the appropriate reference. I know it saves space (and effort) but I think citing the original work is important.

    The reviewer is right; we apologize for falling into this practice. We will reference the original works wherever it is needed.

    Figure 7 is not a model. It is a cartoon depicting what they see with confocal and TEM images.

    We will change the figure; we will include our interpretations of the phenotypes we observed under different experimental manipulations.

    Reviewer #2 (Significance (Required)):

    Overall, this study is one of the first to focus on how the ECM affects coordination of tissue placement. The coordination of tracheal movement with that of the epidermis is very nicely documented here and the observation that the trachea make direct contact with the tendon cells/muscle attachment sites is quite convincing. It is less clear from the data how exactly the cells of the trachea and the ECM are affected by the different perturbations of the ECM. It seems like this could be better done with immunostaining of ECM proteins (collagen-GFP?), cell type markers, and super resolution confocal imaging with combinations of these markers. What happens right at the contact site between the tendon cell and the trachea with the perturbation? I think that at the level of analysis presented here, this study would be most appropriate for a specialized audience working in the ECM or fly embryo development field.

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    Summary The manuscript by Sanchez-Cisneros et al provides a detailed description of the cellular interactions between cells of the Drosophila embryonic trachea and nearby tendon and epidermal cells. The researchers use a combination of genetic experiments, light sheet style live imaging and transmission electron microscopy. The live imaging is particularly clear and detailed, and reveals protruding cells. The results overall suggest that interactions mediated through the ECM contribute to development of trachea and dorsal closure of epidermis. One new aspect is the existence of dorsal trunk filipodia that are under tension and may impact tracheal morphogenesis through required integrin/ECM interactions.

    Major comments:

    • Are the key conclusions convincing? Generally, the key conclusions are well supported by the data, and the movies are very impressive. Interactions between the cell types are clearly shown, as is the correlations in their development. However, some of the images are challenging to decipher for a non-expert in Drosophila trachea, especially the EM images, and some of the data is indirect or a bit weak.

    We thank the reviewer for their observations. As mentioned above in response to Reviewer #1, we will add an overview image of the embryo we processed for TEM that is presented in Figure 2.

    The data related to failure of dorsal closure affecting trachea relies on one homozygous allele of one gene (kayak), and so this is somewhat weak evidence. Even though kay is not detected in trachea, there could be secondary effects of the mutation or another lesion on the mutant chromosome. The segments look a bit uneven in the mutant examples.

    The reviewer is right; as we proposed before, we will complement the kayak experiments with independent approaches that will delay dorsal closure.

    • Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether? Some of the experiments have low n values, especially in imaging experiments, so these may be more preliminary, but they are in concordance with other data.

    The problem we face in our live-imaging experiments is related to the probability of finding the experimental embryos. In most of our experiments we combine double-tissue labelling plus the expression of genetic tools. This generally corresponds to a very small proportion of the progeny. We will aim to have at least 4 embryos per condition.

    • Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation. Higher n-values would substantiate the claims. To strengthen the argument that dorsal closure affects trachea morphogenesis mechanically, the authors might consider using of a combination of kay mutant alleles or other mutant genes in this pathway to provide stronger evidence. Or they could try a rescue experiment in epidermis and trachea separately for the kay mutants.

    We think our experiments delaying dorsal closure using the Gal4/UAS system and a variety of drivers should address the point of the possible indirect effects of kay in tracheal development.

    • Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments. Imaging data can take awhile to obtain, but the genetic experiments could be done in a couple of months, and the authors should be able to obtain any needed lines within a few weeks.

    The reviewer is correct, we will be able to plan our crosses for the proposed experiments within a couple of months.

    • Are the data and the methods presented in such a way that they can be reproduced? Generally, yes. For the deGrad experiments, it is not clear how the fluorescent intensity was normalized - was this against a reference marker?

    Briefly, we used signals from within the embryo as internal controls. In the case of en-gal4, we normalized the signal to the sections of the embryo where en is not expressed and therefore, beta-integrin levels should not be affected. In the case of btl-gal4, we normalized against the signal surrounding the trunks which should also not be affected by the deGradFP system. We will elaborate on these analyses in the methods section.

    Are the experiments adequately replicated and statistical analysis adequate? There are several experiments with low n values, so this could fall below statistical significance. For example, data shown in Fig 1G: n=3; Fig 4D n=4, n=3; Fig 6J n=4

    As mentioned above, we will increase our sample sizes.

    Minor comments:

    • Specific experimental issues that are easily addressable. To make the TEM images more easily interpreted, it would be helpful to provide a fluorescent image of all the relevant cell types (especially trachea, epidermis, muscle, and tendon cells, plus segmental boundaries) labelled accordingly, so that reader can correlate them more easily with the TEM images. They might also include a schematic of an embryo to show where the TEM field of view is.

    We believe this should be addressed by adding the light microscopy section of the embryo with the TEM image overlaid as illustrated above.

    It is hard to be confident that the EM images reflect the cells they claim and that the filopodia are in fact that, at least for people not used to looking at these types of images.

    As we explained in the response to Reviewer #1, we will elaborate on the descriptions of our TEM data. We think that adding the reference micrograph will aid with the interpretations of the TEM images.

    • Are prior studies referenced appropriately? yes

    • Are the text and figures clear and accurate? yes

    • Do you have suggestions that would help the authors improve the presentation of their data and conclusions? The writing could be revised to be a bit clearer. Since the results of the experiments do not support the initial hypothesis, I found it a bit confusing as I read along. It may help to introduce an alterative hypothesis earlier to make the paper more logical and easy to follow. To be more specific, On page 3, the authors say they "show that dorsal trunk displacement is mechanically coupled to the remodelling of the epidermis" and also in the results comment that "With two opposing forces pulling the trunks other factors likely participate in their dorsal displacement, but so far these have remained unstudied." But that doesn't end up being what they find. The results from figure 5 and related interpretation on page 17 says "cell-ECM interactions are important for proper trunk morphology, but not for its displacement." So this was confusing to read and I would encourage the authors to frame the issues a bit differently in terms of tube morphogenesis.

    We see how this might be confusing. We will rewrite the introduction so that the work is easier to follow. To achieve this, we will state from the beginning the mechanisms we anticipate that regulate trunk displacement: 1) adhesion to the epidermis, 2) pulling forces from the dorsal branches and 3) a combination of both.

    Some minor presentation issues: What orientation is the cross-sectional view in figure 1C and movie 1?

    We will add a dotted box that indicates the region that we turned 90° to show the cross-section.

    On page 12, the authors say the "Electron micrographs also suggested high filopodial activity" but activity suggests dynamics that are not clear from EM. This could be re-phrased.

    As the reviewer indicates, we cannot conclude dynamics from a static image. We will replace “suggested high filopodial activity” with “revealed filopodial abundance”.

    Reviewer #3 (Significance (Required)):

    • Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field. The results of the paper are significant in that they characterize a mechanical interaction between two tissue types in development, which are linked by the extracellular matrix that sits between them. It is not clear to me that this describes a "novel mechanism for tissue coordination" as stated in the abstract, but it does characterize this type of interaction in a detailed cellular way.

    • Place the work in the context of the existing literature (provide references, where appropriate). For specialists, the work identifies a novel protruding cell type in the fly embryonic trachea, and provides beautiful and detailed imaging data on tracheal development. The "wavy" trachea phenotype is also uncommon and very interesting, so this result could be linked to the few papers that also describe this phenotype and be built up.

    • State what audience might be interested in and influenced by the reported findings. As it stands, this is most interesting for a specialized audience because it requires some understanding of the development of this system in particular. As it characterizes this to a new level of detail, it could be influential to those in the field. Some addition clarification of the results and re-framing could make the manuscript more clear and interesting for non-specialists.

    • Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate. I work with Drosophila and have studied embryonic and adult cell types, although not trachea specifically. I am familiar with all the genetic techniques and imaging techniques used here.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary

    The manuscript by Sanchez-Cisneros et al provides a detailed description of the cellular interactions between cells of the Drosophila embryonic trachea and nearby tendon and epidermal cells. The researchers use a combination of genetic experiments, light sheet style live imaging and transmission electron microscopy. The live imaging is particularly clear and detailed, and reveals protruding cells. The results overall suggest that interactions mediated through the ECM contribute to development of trachea and dorsal closure of epidermis. One new aspect is the existence of dorsal trunk filipodia that are under tension and may impact tracheal morphogenesis through required integrin/ECM interactions.

    Major comments:

    • Are the key conclusions convincing?

    Generally, the key conclusions are well supported by the data, and the movies are very impressive. Interactions between the cell types are clearly shown, as is the correlations in their development. However, some of the images are challenging to decipher for a non-expert in Drosophila trachea, especially the EM images, and some of the data is indirect or a bit weak.

    The data related to failure of dorsal closure affecting trachea relies on one homozygous allele of one gene (kayak), and so this is somewhat weak evidence. Even though kay is not detected in trachea, there could be secondary effects of the mutation or another lesion on the mutant chromosome. The segments look a bit uneven in the mutant examples.

    • Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

    Some of the experiments have low n values, especially in imaging experiments, so these may be more preliminary, but they are in concordance with other data.

    • Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.

    Higher n-values would substantiate the claims. To strengthen the argument that dorsal closure affects trachea morphogenesis mechanically, the authors might consider using of a combination of kay mutant alleles or other mutant genes in this pathway to provide stronger evidence. Or they could try a rescue experiment in epidermis and trachea separately for the kay mutants.

    • Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.

    Imaging data can take awhile to obtain, but the genetic experiments could be done in a couple of months, and the authors should be able to obtain any needed lines within a few weeks.

    • Are the data and the methods presented in such a way that they can be reproduced?

    Generally, yes. For the deGrad experiments, it is not clear how the fluorescent intensity was normalized - was this against a reference marker?

    • Are the experiments adequately replicated and statistical analysis adequate?

    There are several experiments with low n values, so this could fall below statistical significance. For example, data shown in Fig 1G: n=3; Fig 4D n=4, n=3; Fig 6J n=4

    Minor comments:

    • Specific experimental issues that are easily addressable.

    To make the TEM images more easily interpreted, it would be helpful to provide a fluorescent image of all the relevant cell types (especially trachea, epidermis, muscle, and tendon cells, plus segmental boundaries) labelled accordingly, so that reader can correlate them more easily with the TEM images. They might also include a schematic of an embryo to show where the TEM field of view is.

    It is hard to be confident that the EM images reflect the cells they claim and that the filopodia are in fact that, at least for people not used to looking at these types of images.

    • Are prior studies referenced appropriately?

    yes

    • Are the text and figures clear and accurate?

    yes

    • Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

    The writing could be revised to be a bit clearer. Since the results of the experiments do not support the initial hypothesis, I found it a bit confusing as I read along. It may help to introduce an alterative hypothesis earlier to make the paper more logical and easy to follow. To be more specific, On page 3, the authors say they "show that dorsal trunk displacement is mechanically coupled to the remodelling of the epidermis" and also in the results comment that "With two opposing forces pulling the trunks other factors likely participate in their dorsal displacement, but so far these have remained unstudied." But that doesn't end up being what they find. The results from figure 5 and related interpretation on page 17 says "cell-ECM interactions are important for proper trunk morphology, but not for its displacement." So this was confusing to read and I would encourage the authors to frame the issues a bit differently in terms of tube morphogenesis.

    Some minor presentation issues:

    What orientation is the cross-sectional view in figure 1C and movie 1? On page 12, the authors say the "Electron micrographs also suggested high filopodial activity" but activity suggests dynamics that are not clear from EM. This could be re-phrased.

    Significance

    • Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.

    The results of the paper are significant in that they characterize a mechanical interaction between two tissue types in development, which are linked by the extracellular matrix that sits between them. It is not clear to me that this describes a "novel mechanism for tissue coordination" as stated in the abstract, but it does characterize this type of interaction in a detailed cellular way.

    • Place the work in the context of the existing literature (provide references, where appropriate).

    For specialists, the work identifies a novel protruding cell type in the fly embryonic trachea, and provides beautiful and detailed imaging data on tracheal development. The "wavy" trachea phenotype is also uncommon and very interesting, so this result could be linked to the few papers that also describe this phenotype and be built up.

    • State what audience might be interested in and influenced by the reported findings.

    As it stands, this is most interesting for a specialized audience because it requires some understanding of the development of this system in particular. As it characterizes this to a new level of detail, it could be influential to those in the field. Some addition clarification of the results and re-framing could make the manuscript more clear and interesting for non-specialists.

    • Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

    I work with Drosophila and have studied embryonic and adult cell types, although not trachea specifically. I am familiar with all the genetic techniques and imaging techniques used here.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary: In this paper, the authors explore the relationships between two Drosophila tissues - the epidermis and tracheal dorsal trunk (DT) - that get dorsally displaced during mid-late embryogenesis. The show a nice temporal correlation between the movements of the epithelia during dorsal closure and DT displacement. They also show a correlation between the movement of an endogenously tagged version of collagen and the DT, suggesting that the ECM may contribute to this coordinated movement. Through high magnification TEM, they show that tracheal cells make direct contact with the subset of epithelial cells, known as tendon cells, that also serve as muscle attachment sites. In between these contact sites, tracheae are separated from the epithelia by the muscles. Furthermore, the TEMs and confocal imaging of tracheal cells expressing a membrane marker at these contact sites show that the tracheal cells are extending filopodia toward the tendon cells. The authors then explore how a variety of perturbations to the ECM produced by the tendon and DT cells affect DT and epithelial movement. They find that expressing membrane-associated matrix metalloproteases (MMP1 or MMP2) in tendon cells as well as perturbations in integrin or integrin signaling components leads to delays in dorsal displacement as well as defective lengthening of the tracheal DT tubes. They find that defects in the association between the tracheal and epidermal ECM attachments affect dorsal displacement of the epidermis, disrupting dorsal closure.

    Major comments: I like the goals of this paper testing the idea that the ECM plays important roles in the coordination of tissue placement, and I think they have good evidence of that from this study. However, I disagree with the conclusions of the authors that disrupting contact between DT and the tendon cells has no effect on DT dorsal displacement. DT tracheal positioning is clearly delayed; the fact that it takes a lot longer indicates that the ECM does affect the process. It's just that there are likely backup systems in place - clearly not as good since the tracheal tubes end up being the wrong length. It also seems important that the parts of the DT where the dorsal branches (DB) emanate are moving dorsally ahead of the intervening portions of the trachea. This suggests to me that the DB normally does contribute to DT dorsal displacement and that this activity may be what helps the DT eventually get into its final position. The authors should test whether the portions of the DT that contact the DB are under tension. If the DB migration is providing some dorsal pulling force on the DT, this may also contribute to the observed increases in DT length observed with the perturbations of the ECM between the tendon cells and the trachea - if tube lengthening is a consequence of the pulling forces that would be created by parts of the trachea moving dorsally ahead of the other parts. Here again, it would be good to test if the DT itself is under additional tension when the ECM is disrupted.

    Minor comments: The authors need to do a much better job in the intro and in the discussion of citing the work of the people who made many of the original findings that are relevant to this study. Many citations are missing (especially in the introduction) or the authors cite their own review (which most people will not have read) for almost everything (especially in the discussion). This fails to give credit to decades of work by many other groups and makes it necessary for someone who would want to see the original work to first consult the review before they can find the appropriate reference. I know it saves space (and effort) but I think citing the original work is important.

    Figure 7 is not a model. It is a cartoon depicting what they see with confocal and TEM images.

    Significance

    Overall, this study is one of the first to focus on how the ECM affects coordination of tissue placement. The coordination of tracheal movement with that of the epidermis is very nicely documented here and the observation that the trachea make direct contact with the tendon cells/muscle attachment sites is quite convincing. It is less clear from the data how exactly the cells of the trachea and the ECM are affected by the different perturbations of the ECM. It seems like this could be better done with immunostaining of ECM proteins (collagen-GFP?), cell type markers, and super resolution confocal imaging with combinations of these markers. What happens right at the contact site between the tendon cell and the trachea with the perturbation? I think that at the level of analysis presented here, this study would be most appropriate for a specialized audience working in the ECM or fly embryo development field.

  4. Review Commons

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    Referee #1

    Evidence, reproducibility and clarity

    In this manuscript, Sanchez-Cisneros and colleagues, examine how tracheal cell adhesion to the ECM underneath the epidermis helps shape the tracheal system. They show that if cell-ECM adhesion is perturbed the development of the tracheal system and the epidermis is disrupted. They also detect protrusions extending from the dorsal trunk cells towards the ECM.

    The work is novel, the figures are clear, and the questions are well addressed. However, I find that some of the claims are not completely supported by the data presented. I have some suggestions that will, I believe, clarify certain points.

    Major comments

    At the beginning of the results section as in the introduction the authors claim that "It is generally assumed that trunk displacement occurs due to tip cells pulling on the trunks so that they follow their path dorsally." This sentence is not referenced, and I do not know where it has been shown or proposed to be like this. In addition, the comparison with the ventral branches is also not referenced and the movie does not really show this. Forces generated by tracheal branch migration have been shown to drive intercalation (Caussinus E, Colombelli J, Affolter M. Tip-cell migration controls stalk-cell intercalation during Drosophila tracheal tube elongation. Curr Biol. 2008;18(22):1727-1734. doi:10.1016/j.cub.2008.10.062), but not dorsal trunk (DT) displacement. However, to rule out the possibility that DT displacement and the phenotype observed in XXX is due to dorsal branch pulling forces, the authors should analyze what happens in the absence of dorsal branches (in condition of Dpp signalling inhibition as in punt mutants or Dad overexpression conditions).

    I am concerned about the TEM observations. The authors claim they can identify tracheal cells by their lumen (Fig. 2 C'). However, at stage 15, the tracheal lumen should be clearly identifiable, and the interluminal DT space should be wider relative to the size of the cells. In this case, there is nothing telling us that we are not looking at a dorsal branch or lateral trunk cell. Furthermore, at embryonic stage 15, the tracheal lumen is filled with a chitin filament, which is not visible in these micrographs. Also, there is quite a lot of tissue detachment and empty spaces between cells, which might be a sign of problems in sample fixing. Better images and more accurate identification of dorsal trunk cells is necessary to support the claim that "These experiments revealed a novel anatomical contact between the epidermis and tracheal trunks".

    Timelapse imaging of the protrusions in DT cells is done with frames every 4 minutes (Video S3). This is not enough to properly show cellular protrusions and the images do not really show interaction with the epidermis. Video S4 has a better time resolution but it is very short and only shows the cut moment. Video S4, shows the cut, but the reported (and quantified recoil) is not clear. Nevertheless, the results are noteworthy and should be further analysed. Provided these embryos survive, would it be possible to check if embryos after laser cutting will develop wavy DTs?

    What happens to the larvae under the genetic conditions presented in Fig.S3? Do they reach pupal stages? Do these animals reach adult stages?

    The kayak phenotypes are very interesting and perhaps the authors could explore them more. As in inhibition of adhesion to the ECM, kay mutants display wavy dorsal trunks. Do they have defective adhesion? Fos being a transcription factor, this is a possibility. The authors should at least discuss the kay phenotypes more extensively and present a suitable hypothesis for the phenotype.

    Minor comments

    Page 2 Line 9/10 The sentence "tracheal tubes branch and migrate over neighbouring tissues of different biochemical and mechanical properties to ventilate them." should be rewritten. Tracheal cells do not migrate over other tissues to ventilate them.

    Page 2 Line 24/25 The sentence "It has been generally assumed that trunks reach the dorsal side of the embryo because of the pulling forces of dorsal branch migration." needs to be backed up by a reference.

    Page 7 Line 32/23 In this sentence, the references are not related to dorsal closure "Similarly, the signals that regulate epidermal dorsal closure do not participate in tracheal development, or vice versa (Letizia et al., 2023; Reichman-Fried et al., 1994)."

    Page 12 Line 1 "Muscles attach to epidermal tendon cells through a dense meshwork of ECM" this sentence must be referenced.

    Fig. S1- Single channel images (A'-C' and A'-C') should be presented in grayscale.

    Fig. S4- Single channel images (A'-D' and A'-D') should be presented in grayscale.

    Significance

    The findings shown in this manuscript shed light on the interactions and cooperation between two organs, the tracheal system and the epidermis. These interactions are mediated by cell-ECM contacts which are important for the correct morphogenesis of both systems. The strengths of the work lie on its novelty and live analysis of these interactions. However, its weaknesses are related to some claims not completely backed by the data, some technical issues regarding imaging and some over-interpreted conclusions.

    This basic research work will be of interest to a broad cell and developmental biology community as they provide a functional advance on the importance of cell-ECM interactions for the morphogenesis of a tubular organ. It is of specific interest to the specialized field of tubulogenesis and tracheal morphogenesis.

  5. Version published to 10.1101/2025.03.15.643376 on bioRxiv