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Self-organized patterning of cell morphology via mechanosensitive feedback

Natalie A. Dye, Marko Popovic, K. Venkatesan Iyer, Suzanne Eaton, Frank Julicher

Preprint posted on April 18, 2020 https://www.biorxiv.org/content/10.1101/2020.04.16.044883v1

A game of tug of war: cell morphology and mechanosensitive feedback.

Selected by Mariana De Niz

Background

In the process of morphogenesis, tissues with complex morphologies are formed due to the interplay between various cells. Ultimately, tissue organization is characterized by specific patterns of cell morphology. These patterns are important, and in many cases are directly related to the function of the resulting tissue. Likewise, spatial patterns of cell morphology can guide the morphogenetic process itself. How such patterns emerge in developing tissues is a fundamental open question. In their work, Dye and colleagues investigate the emergence of tissue-scale patterns of cell morphology and mechanical tissue stress in the Drosophila larval wing imaginal disc, which has a geometry suitable for studying spatial patterns of cell morphology (1).

Figure 1. Cell morphology patterns can persist and strengthen in the absence of differential growth. (A) Wing disc growing in ex vivo culture. Analysis of apical cell morphology in the proliferating disc proper layer in a 2D projection. Spatial maps of cell area (B) or cell elongation (C). (Figure obtained from Ref 1).

Key findings and developments

            In their work, the authors imaged and quantified the spatial patterns of cell morphology, cell divisions, and cell rearrangements during the middle of the third Drosophila larval instar. They used live microscopy to perform dynamic imaging of growing explanted wing discs. This allowed observation of a pattern of tangential cell elongation, with cells elongating perpendicular to the radial axis. This pattern was interrupted around the dorso-ventral boundary, where cells instead elongate parallel to the boundary. Altogether, the spatial maps of cell morphology, allowed identification of radial gradients in cell area and cell elongation existing outside of the dorso-ventral boundary region, and showed that these cell morphology patterns persist and strengthen in time in the absence of differential growth.

            Following these observations, the authors went on to analyse spatial patterns in cellular contributions to tissue shear –  the latter of which is affected by contributions from cell divisions, cell elongation changes, T1 transitions, and correlation effects (arising from cell elongation and cell rotation). They determined the magnitude of radial and tangential patterns, and found that while the radial component of tissue shear was small, there was a pronounced build-up of tangential patterns of cell elongation, accompanied by a radial pattern of T1 transitions*, and correlation effects. (*T1 transitions consist of a phase characterized by the collapse of a junction with dorso-ventral orientation, and another phase characterized by expansion of a new junction in anterior-posterior orientation).

            The authors then used a biophysical model to explore whether radially patterned T1 transitions could account for the observed cell morphology patterns in the wing disc. The model was adapted to the tissue material properties. The main finding was that the steady state elongation pattern in the wing disc, may largely result from polarity-driven cell rearrangements. Further exploration of tissue stress and mechanical parameters of the models was performed using laser ablation, whereby the authors performed circular cuts to analyse the final relaxed position of the inner and outer elliptical contours of tissue formed by the cut – and called this method ESCA (elliptical shape after circular ablation). This allowed inference of anisotropic (direction dependent properties) and isotropic (direction-independent properties) tissue stress, and polarity-driven stress. The main finding was that the average cell elongation correlates with the direction of shear stress, but that cells around the dorso-ventral boundary have different mechanical properties than elsewhere in the tissue, with cells elongating less when subject to comparable amounts of stress. Outside this region, the stress profile in the wing disc showed that polarity-driven stress is significant, and that pressure increases towards the centre.

            Having identified this stress profile, the next step was to identify the radial orientational cue to generate the observed patterns. For this, the authors generated knockdown mutants lacking important components of well-characterized pathways related to planar cell polarity (PCP), and analysed cell elongation patterns after their removal. None of the knockdowns showed reduced tangential cell elongation. This led the authors to instead considered mechanical stresses in the tissue, as another potential factor influencing polarity. They found that introducing mechanosensitive feedback to the model gave rise to spontaneous emergence of a cell polarity cue, by self-organization. The model allowed hypothesizing that the mechanosensitive model can account for the radial pattern of cell morphology in the wing disc. To test the hypothesis, the authors generated knock-downs of Myosin VI, a molecular motor implicated in mechanosignaling, which reorganizes the actin-myosin cytoskeleton in response to mechanical stress. The knockdown showed a reduction in the magnitude of tangential cell elongation, and a correspondingly increased cell area in the central part of the wing. The overall conclusion was that suppression of mechano-sensitivity weakens the gradients in cell elongation and cell size.

 

 What I like about this preprint

I like this preprint because I firmly believe that science benefits from inter-disciplinarity, and this work explores the question of tissue morphology, from a biophysical point of view. Furthermore, the authors interestingly explore and incorporate existing tools to answer novel questions with this multi-faceted angle in mind. Moreover, I like that the questions arising from, and addressed in this preprint are very clear and structured. I also like the outstanding questions arising from this- which I certainly find interesting for various fields of research (beyond developmental biology and Drosophila -specific studies).

 

References

  1. Dye NA, Popovic M, Iyer KV, Eaton S, Julicher F, Self-organized patterning of cell morphology via mechanosensitive feedback, bioRxiv, 2020
  2. Acharya, B. R., Nestor-Bergmann, A., Liang, X., Gupta, S., Duszyc, K., Gauquelin, E., Gomez, G. A., Budnar, S., Marcq, P., Jensen, O. E., Bryant, Z., & Yap, A. S. A Mechanosensitive RhoA Pathway that Protects Epithelia against Acute Tensile Stress. Developmental Cell, 47(4), 439- 452.e6. 2018.

 

Posted on: 22nd May 2020

doi: https://doi.org/10.1242/prelights.21043

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  • Author's response

    Natalie A. Dye, Marko Popovic, Frank Julicher shared

    Open questions 

    1.Firstly, I liked this study very much, and it appears it would be very relevant to various fields of research. I was wondering whether (and how) your key findings in this Drosophila work, are conserved in other cells, tissues and organisms?

    The theory is certainly general, and we think it is very likely that it applies to other systems. The beauty of the Drosophila wing is that it is so amenable to microscopy, and thus the patterns of cell size and shape are relatively easy to visualize and quantify. But there is nothing in our model that is Drosophila-specific – it postulates that a feedback between stress and polarity can lead to self-organized morphological patterning. In other systems, there are many examples of cytoskeleton polarizing in response to stress, and it need not be that the same pathway mediates the mechanosensitivity underlying self-organized patterning of morphology in every system. We intend to explore this topic in future work.

    2. You mention throughout your work, the differences observed in the dorso-ventral boundary. What is the relevance of these differences? Is it known whether and how the DV boundary affects wing function? and are you planning to explore in equal detail, questions related to this location too?

    To our knowledge, this is the first work that observes any evidence that the DV boundary is different than the rest of the wing pouch, with regard to cell morphology and mechanical properties. Thus, it is an important result to report, although we did not have the space to fully explore the underlying mechanism or the importance of this result here. One of the directions we would like to explore in the future is how the juxtaposition of two regions of different mechanical properties may affect the self-organization behavior we observe. Here, we only considered a homogenous, radially-symmetric case. In reality, the wing is a bit more complicated because of the DV boundary, and later stages also have similar disruptions at the AP boundary. Thus, it is an important future direction to understand the evolution of these more complex patterns, using both theory and experiment. The genetic tractability of the Drosophila wing will allow us to both destroy and expand these regions by altering the signaling systems that are known to delineate these regions (ie., Wingless/Notch at the DV boundary).

    3.How do you hypothesize that disruption of mechanical feedback, and the resulting changes in cell morphology and cell patterns, would be related to disease? Are there well-characterized diseases known to arise from mechano-sensing disruptions?

    Interesting question, and I can only speculate for now. There are many examples of diseases resulting from loss of mechanosensation. There are MyoVI mutants, for example, that are associated with deafness. However, it is hard to say how many of these diseases result from the loss of the kind of self-organized morphological patterning we observe in the fly wing. I think answering your earlier question of how well conserved this mechanism is in other tissues/organisms will be a good first step toward understanding how perturbations may lead to disease.

    4.In this work you specifically investigated MyoVI as a key molecular player. Two questions arising are a) how does MyoVI contribute to the actin-myosin cytoskeleton in a cell, and b) are there other players you have in mind, equally relevant to mechano-sensing (as it is probably unlikely that only MyoVI controls this key phenomenon)?

    Yes, there is certainly a lot more to do to understand MyoVI and mechanosensation in general in this system. The Acharya et al (Dev Cell 2018) paper that we reference characterized a MyoVI-dependent mechanosensation pathway in detail using a mammalian cell culture model. They showed that MyoVI initiates a cascade of signaling going through RhoGTPase to affect the organization of the actin-myosin cytoskeleton. That work was the inspiration for trying the MyoVI RNAi in Drosophila. You are correct that there are other known mechanosensing elements, so I was actually quite impressed that we saw a phenotype at all upon perturbation of just MyoVI. It is possible, however, that there are different pathways activated for different types or magnitudes of stresses. While we focused in this paper on the tissue scale, using continuum models, it is certainly an important and interesting future direction to address the underlying molecular and cellular details contributing to this self-organized behavior.

    5.Although you investigated key molecules in the PCP pathway, and observed that knockdown mutants weren’t significantly altered, is it possible that overall patterning of cell morphology is the result of contributions from these pathways as well (although they might not be equally central as others)?

    We do notice some subtle differences upon perturbation of PCP pathways. For example, the DV boundary is somehow less pronounced upon removal of Fat and Dachs. Also, removal of either PCP pathway does seem to emphasize the tangential cell elongation pattern (cells are even more elongated than in the wild type). However, we were interested in identifying the cue that patterns radial cell rearrangements, and removal of such cue would weaken the radial cell elongation instead. Thus, the data we have does not indicate that either PCP pathway is the radial cue that patterns the cell rearrangements. Nonetheless, the subtle phenotypes we observe may indicate that the PCP pathways otherwise affect the mechanical properties or dynamic cell behavior. More experiments, such as laser ablations in the PCP mutants, would be required to investigate this possibility further.

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