Constriction forces imposed by basement membranes regulate developmental cell migration

  1. Ester Molina López
  2. Anna Kabanova
  3. Maria D. Martín-Bermudo

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Abstract

The basement membrane (BM) is a specialized extracellular matrix, which underlies or encase developing tissues. Mechanical properties of encasing BMs have been shown to profoundly influence the shaping of associated tissues. Here, we use the migration of the border cells (BCs) of the Drosophila egg chamber to unravel a new role of encasing BMs in developmental cell migration. BCs move between a group of cells, the nurse cells (NCs), that are enclosed by a monolayer of follicle cells (FCs), enveloped in turn by a BM, the follicle BM. We show that increasing or reducing the stiffness of the follicle BM, by altering laminins or Coll IV levels, conversely affects BC migration speed and alters migration mode and dynamics. Follicle BM stiffness also controls pairwise NC and FC cortical tension. We propose that constriction forces imposed by the follicle BM influence NC and FC cortical tension, which, in turn, regulate BC migration. Encasing BMs emerge as key players in the regulation of collective cell migration during morphogenesis.

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    Reply to the reviewers

    'The authors do not wish to provide a response at this time.'

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    Referee #3

    Evidence, reproducibility and clarity

    Summary: The manuscript analyzes how the constriction of a tissue by an enveloping basement membrane alters the migration of cells migrating through that tissue. The tissue analyzed is the Drosophila egg chamber, an important model for basement membrane studies in vivo, and the cells migrating through it are the border cells. The border cells migrate through the center of the egg chamber, moving as a cluster between the nurse cells, which are in turn surrounded by follicle cells, which secrete the basement membrane on the outside of the egg chamber. The authors decrease and increase the basement membrane stiffness with various genetic perturbations, and they find that the border cells move more rapidly when the stiffness is reduced. They then investigate how basement membrane stiffness is communicated to the border cells several cell layers inside, by measuring cortical tension with laser-recoil. They found that external basement membrane stiffness alters the cortical tension of the nurse cells and the follicle cells, such that reduced matrix stiffness causes reduced cortical tension; further, reducing cortical tension directly within the cells also results in increased border cell migration rates. They conclude that basement membrane stiffness can alter cell migration in a new way, by altering constriction and cortical tension, with an inverse relationship between stiffness and migration rate. This is a strong manuscript and I would request very few changes.

    The authors are commended on the rigor and completeness of their study. Several independent methods are used to alter basement membrane stiffness (loss of laminin, knock-down of laminin, knock-down of collagen IV, over-production of collagen IV - all of which end up changing collagen IV levels) and all show the same result. Further, they are extremely rigorous about testing and excluding an attractive alternative hypothesis, that the basement membrane of the border cell cluster itself controls its migration rate. The use of mirror-Gal4 is very elegant and convincing, as it expressed only in the central part of the egg chamber, and they found border cells responded differently only in that region. Moreover, the authors were exceptionally thorough in reproducing the basement membrane mechanical data in their own hands using the bursting assay. Overall, the experimental data support the claims of the paper. There is only one more control I would like to see, for the knockdown of laminin in the border cell cluster with a triple-Gal4 combination. Presumably using all three Gal4 lines was necessary to get complete knockdown, and it would be nice to see anti-laminin for the border cell cluster under these knockdown conditions.

    Despite the rigor, because all of the manipulations to the basement membrane alter the levels of collagen IV, the authors cannot formally exclude the possibility that collagen IV in the basement membrane has another function besides stiffness, perhaps sequestering a signaling ligand, and that this other function somehow alters the cortical tension of the egg chamber. In the paper by Crest et al, externally applied collagenase served as a control for this possibility, but collagenase will not work for the authors because this study is in vivo. I suggest the authors bring up this caveat in the discussion. If they wanted to extend the study (optional), they could knock down the crosslinking enzyme peroxidasin in the egg chamber, which ought to reduce basement membrane stiffness without changing the collagen content. The problem here is that it hasn't already been shown to work that way in the egg chamber, and so both stiffness and collagen levels would need to be measured. Testing the stiffness directly would be difficult, since the bursting assay is not actually a measurement of stiffness (more on that below). Rather than go this route, I suggest just acknowledging the formal possibility, which seems to me unlikely anyway.

    In terms of clarity, the manuscript absolutely needs a schematic at the beginning to introduce the egg chamber and border cell migration, labeling the cell types, showing the route and direction of border cell migration, and labeling the A/P axis. Without this the non-expert reader cannot readily understand the study.

    Finally, in terms of clarity, the authors repeatedly use statements such as "stiffness influences migration rate". Influences how? These results are not intuitive to me, and it would help enormously if the authors would make statements like, decreasing stiffness increases migration (as I tried to in my summary). Here are two examples of statements to refine: • Line 189 - "We found that reducing laminin levels affected the migration speed of both phases (Fig.1F, G)." Please say increased, not affected. • Line 245 -"Altogether, these results demonstrate that the stiffness of the follicle BM influences dynamics and mode of BC migration." Again, be specific about how. There are many such statements, from the abstract to the results to the discussion, where it would help the clarity to be more precise about what kind of influence.

    Minor comments: • The movies are beautiful! • All the quantitative data are shown in bar charts with means and errors. It is much better to show the individual data points, superimposing the means and distributions on top of the individual points. • The bursting assay does not actually measure basement membrane stiffness; rather, it measures failure after elastic expansion. These are related, as was found by Crest et al and the authors say that at one point, but stiffness and failure are not the same thing. Please change the language discussing this assay to "mechanical properties" rather than stiffness. • The laser-recoil assays are done well and are convincing. Throughout the results section, the authors describe these as measuring "cortical tension", which is correct. However, in the figure legends the language changes to "membrane tension" which is only one component of cortical tension. Change them all to cortical tension. • In the Discussion, it would be nice to include something on the two different modes of migration (tumbling and not tumbling). • I suggest changing the title to remove the word "forces", because forces are never directly measured from basement membrane. • Although Dai et al (Science 2020) is discussed near the end, I suggest bringing this reference up to the introduction, so the reader can have the background on the mechanical aspects of border cell migration at the start of this study. • Two typos (there may be more): At the bottom of Fig. 2, text turns strangely white that should probably be black; and in line 260, you mean Fig. S5 not S4 (laser ablation).

    Significance

    Mechanobiology, and mechanobiology of the basement membrane, is a vibrant area of study now, arising from the intersection of biophysics/engineering and genetics. There is general interest in how the basement membrane alters forces within the tissue, and this study is the first to my knowledge to relate basement membrane mechanics to migration via constriction and cortical tension. The authors do a great job of discussing the broader significance of their work in the Discussion. To greatly broaden the scope of this work in the future, the authors could collaborate with a mouse team to look for similar responses in a mammalian tissue, as they discuss. It is worth noting that there is a lot of work on matrix stiffness and migration showing that stiffness promotes migration speed; in these cases, matrix is a substrate, not a compression mechanism. But the opposite nature of the result in interesting and makes this work non-intutive and perhaps hard for some readers to grasp.
    As the paper is written now, I think the audience for this work would mostly be oogenesis, border cell migration, and/or basement membrane researchers in the Drosophila community, of which there are many (I am in this camp). With some rewriting to make it more accessible to other audiences, I think it would be interesting to a larger developmental biology audience. The content is not like any other paper I know, but it may be similar in scope and subject matter to the papers detailing how follicle cells and basement membrane interact during follicle rotation.

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    Referee #2

    Evidence, reproducibility and clarity

    Ester and her colleagues described a force model that controls border cell migration by varying the stiffness of the basement membrane. It's based on the modification of laminin and Coll IV which are components of the basement membrane. To reduce BMs stiffness, they introduced LanB1RNAi or the LanB1 mutant to inhibit laminin production or vkgRNAi to reduce Coll IV. They also applied EHBP1mCh to enhance BM stiffness. Furthermore, they applied laser ablition to confirm that the BM stiffness affects the tension of nurse cells and follicle cells, thus regulating border cell behavour by changing environmental properties. It is a nice work revealing how the environment controls border cell migration; however, there are several points that concern me:

    1. It's reported that actin polymerization at the front of the cell generates protrusions, as well as that myosin contractility helps to suppress lateral random protrusions, thus leading to a directed and efficient cell migration. So why do more lateral protrusions (tj>LanB1RNAi) produce a faster migration speed?
    2. We know some labs also did experiments with those Kel/Dic mutant flies. And the Kel mutant is very sick, which sometimes leads to NC degeneration. As a result, we have serious doubts that this mutant's border cell migration will remain normal.
    3. From figure4, we noticed that with mirrGal4, the vertex distance increase is much lower than tjGal4 (control of D, H and K), and even with expressing the EHPB1mCh, the distance is still lower than the tjGal4 control. These indicate the NC cortical tension is lower with mirrGal4 expression, which is patially against the paper's main point. (Similar issue in figure5 D and E).
    4. Sfigure1 A and B seem not to have the right contrast (the blue and the red should have the same brightness), so the comparison of the intensities might be inaccurate and needs to be requantified after adjustment of the images.
    5. Sfigure2 A-E showed that the vkgRNAi has the highest bursting frequency, whereas F and G do not. And the majority of the data from F does not fit with A-E, and it is unclear what timepoint sF.G. is at.
    6. SFigure 6 only displayed a representative image of the control condition; the lack of representative images for the other conditions resulted in unconvincing results.
    7. Some figures and movies have prominent variation of migrating stages, such as not-detached border cells compared with detached border cells. This might strongly cause the results inconsistent with each other.
    8. There are numerous typos in both the manuscript and the figures. Based on all these concerns, I recommend authors to do some improvement before this manuscript is accepted by some reputed journals.

    Significance

    Strengths: the manuscript is well written and organised; limitation: figures and results are not supportive enough, and thus conclusion is not completely convincing, statistical quantification is not clear and somehow confusing.

    Advice: If the conclusion is solid, this story will fill the unclear importance of surrounding environment on cell-rich tissue for collective cell migration. Concept is very novel while needing more supporting data. It is a fundamental study for development biology.

    Audience: The story will fit well for developmental and cell biology, as well as people with biomechanical background.

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    Referee #1

    Evidence, reproducibility and clarity

    The manuscript by Lopez et al have employed the ex vivo model of Border cell migration in Drosophila ovaries to examine the constriction forces imposed by basement membrane on migrating border cells. The authors have extensively employed live cell imaging coupled with genetics to demonstrate that basement membrane encasing fly eggs modulates the dynamics of migrating border cells. Through laser ablation experiments they show that basement influences the tension of the underlying follicle cell and nurse cells which in turn affects the migration efficiency of collectively moving border cells. Over all the experiments are well quantified with good degree of statistics that drive the claim of the authors.

    Major comments: Over in all the images, it is very hard to appreciate the overall contour of the egg chamber. This is important to get an insight regarding the stage of the egg chamber being evaluated.

    1. By depleting the constituents of basement membrane, the authors show that the speed of the migrating border cells increases. However in Supplementary Figure 1A and B where that authors have depleted LanB1, the migrating border cell cluster seems to lag while the control has reached the oocyte boundary. Is this a single off phenomena?

    2. This is regarding the osmotic swelling experiments. The frequency and speed of bursting of egg chambers in deionized water was used to evaluate the stiffness of basement membrane in different genetic background. As egg chambers of different stages have variable sizes, it would be fair to evaluate egg chambers of only a particular stage for this analysis as the tonocity of the egg chambers may depend on their size.

    3. Line 211, "Live time lapse imaging, showed that the overexpression of EHBP1mCh in all FCs delayed BC migration (tslGFP; tj> EHBP1mCh, Figure S4A-B', Movie S4, n=6)." Though the border cell cluster hasn't moved significantly in Fig S4B', the egg chamber development seems to be stalled as the movement of main body follicle cells is affected. My concern if over expression of EHBP1mCh in the follicle cells is stalling the oogenesis itself could that indirectly affect the border cell movement. Secondly though EHBP1 has been shown to affect secretion of the basement membrane constituents, it could also modulate asymmetric secretion of other components. Can the authors evaluate if over expression of EHBP1mCh rescues the delay in migrating border cells in Lanb1 heterozygous background to render stronger support to their claim.

    4. In Supplementary Fig 7 B and B' the nurse cell morphology seems to be affected. Could the distorted nurse cell morphology in the abi-depleted germline cell affecting the migration efficiency of border cells.

    5. Line 313-314 The authors state that "The radius of curvature of a spherical interface is inversely proportional to the difference in pressure between the two sides of the interface." This may be applicable to a smooth surface but may be not directly applicable to the cell membrane as there are local regional variations and thus any inference on the cytoplasmic pressure of nurse cells may be misleading.

    Minor comment:

    1. In supplementary figure 6D, the square boxes are obscuring the border cell membrane and it will be better if the authors can modify the figure to render more clarity.
    2. There are couple of places where sentence structure needs to be corrected.

    Referees cross-commenting

    I agree with all the comments of other reviewers. Overall I also feel that results do not strongly support the main conclusions. The authors draw major conclusions based on experiments that are merely suggestive rather than being conclusive. Some of the concerns are listed. Like Reviewer 3 raising the concern that Collagen IV may have other functions in the basement membrane other than providing stiffness. A similar concern I too have raised regarding over-expression of EHBP1. I agree with Reviewer 3 that there are several other factors that can affect the outcome of bursting assay besides the stiffness of the basement membrane itself. So the authors need to be careful in linking the bursting frequency of the egg chamber with the stiffness of the basement membrane itself.

    I agree with other reviewers that the quality of the images need to be better. In addition, the image presented should be representative of the population and should fit with the over claim made by the authors (Point No 3 of Reviewer 2 and Point No 1 of Reviewer1). I also agree that authors need to explain Reviewer 2's concern (Point No-1) as to why the lateral protrusion in tj>LanB1RNAi doesn't inhibit the movement of border cell clusters but rather produce faster migration speeds?

    Lastly it is important for the authors to verify that like Kel/Dic mutants are indeed effective or any genetic perturbation like overexpression of EHBP1mCh is not the stalling the oogenesis progression perse, thus giving a false impression of altered migratory speed of border cell clusters.

    Significance

    The role basement membrane is well documented in affecting the shape of neighbouring cells Here the authors claim that the stiffness of basement membrane is regulating the migration efficiency of the border cells. I believe that basement membrane encasing the follicle and underlying germline cells provides a very narrow passage for the border cells to migrate. Any mechanical perturbation that releases or increases the pressure or make the nurse cells membranes less or more taut will affect the dynamics of migrating border cells. Though the authors have demonstrated this with very elegant experiments, I am afraid that their findings are standard outcomes in any physically constrained system and somehow doesn't significantly advance the field.

  5. Version published to 10.1101/2022.12.19.520983 on bioRxiv