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The actin cable is dispensable in directing dorsal closure dynamics but neutralizes mechanical stress to prevent scarring in the Drosophila embryo

Nature Cell Biology volume 18, pages 1149–1160 (2016)Cite this article

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Abstract

The actin cable is a supracellular structure that embryonic epithelia produce to close gaps. However, the action of the cable remains debated. Here, we address the function of the cable using Drosophila dorsal closure, a paradigm to understand wound healing. First, we show that the actin cytoskeleton protein Zasp52 is specifically required for actin cable formation. Next, we used Zasp52 loss of function to dissect the mechanism of action of the cable. Surprisingly, closure dynamics are perfect in Zasp52 mutants: the cable is therefore dispensable for closure, even in the absence of the amnioserosa. Conversely, we observed that the cable protects cellular geometries from robust morphogenetic forces that otherwise interfere with closure: the absence of cable results in defects in epithelial organization that lead to morphogenetic scarring. We propose that the cable prevents morphogenetic scarring by stabilizing cellular interactions rather than by acting on closure dynamics.

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Figure 1: The actin cable is a discontinuous structure.
Figure 2: Zasp52 in an upstream regulator of actin cable formation.
Figure 3: The actin cable is dispensable and does not provide a major contractile force during dorsal closure.
Figure 4: The actin cable is dispensable for embryonic wound healing.
Figure 5: The actin cable is crucial for leading edge straightness.
Figure 6: The actin cable maintains local tension to homogenize leading edge behaviour.
Figure 7: The actin cable prevents ectopic canthi formation.
Figure 8: The actin cable prevents morphogenetic scarring.

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References

  1. Wood, W. et al. Wound healing recapitulates morphogenesis in Drosophila embryos. Nat. Cell Biol. 4, 907–912 (2002).

    Article  CAS  Google Scholar 

  2. Martin, P. & Parkhurst, S. M. Parallels between tissue repair and embryo morphogenesis. Development 131, 3021–3034 (2004).

    Article  CAS  Google Scholar 

  3. Belacortu, Y. & Paricio, N. Drosophila as a model of wound healing and tissue regeneration in vertebrates. Dev. Dyn. 240, 2379–2404 (2011).

    Article  CAS  Google Scholar 

  4. Brock, J., Midwinter, K., Lewis, J. & Martin, P. Healing of incisional wounds in the embryonic chick wing bud: characterization of the actin purse-string and demonstration of a requirement for Rho activation. J. Cell Biol. 135, 1097–1107 (1996).

    Article  CAS  Google Scholar 

  5. Martin, P. & Lewis, J. Actin cables and epidermal movement in embryonic wound healing. Nature 360, 179–183 (1992).

    Article  CAS  Google Scholar 

  6. Davidson, L. A., Ezin, A. M. & Keller, R. Embryonic wound healing by apical contraction and ingression in Xenopus laevis. Cell Motil. Cytoskeleton 53, 163–176 (2002).

    Article  Google Scholar 

  7. Mateus, R. et al. In vivo cell and tissue dynamics underlying zebrafish fin fold regeneration. PLoS ONE 7, e51766 (2012).

    Article  CAS  Google Scholar 

  8. Bement, W. M., Forscher, P. & Mooseker, M. S. A novel cytoskeletal structure involved in purse string wound closure and cell polarity maintenance. J. Cell Biol. 121, 565–578 (1993).

    Article  CAS  Google Scholar 

  9. Danjo, Y. & Gipson, I. K. Actin ‘purse string’ filaments are anchored by E-cadherin-mediated adherens junctions at the leading edge of the epithelial wound, providing coordinated cell movement. J. Cell Sci. 111, 3323–3332 (1998).

    CAS  PubMed  Google Scholar 

  10. Jacinto, A., Martinez-Arias, A. & Martin, P. Mechanisms of epithelial fusion and repair. Nat. Cell Biol. 3, E117–E123 (2001).

    Article  CAS  Google Scholar 

  11. Young, P. E., Richman, A. M., Ketchum, A. S. & Kiehart, D. P. Morphogenesis in Drosophila requires nonmuscle myosin heavy chain function. Genes Dev. 7, 29–41 (1993).

    Article  CAS  Google Scholar 

  12. Jacinto, A. et al. Dynamic actin-based epithelial adhesion and cell matching during Drosophila dorsal closure. Curr. Biol. 10, 1420–1426 (2000).

    Article  CAS  Google Scholar 

  13. Jacinto, A. et al. Dynamic analysis of actin cable function during Drosophila dorsal closure. Curr. Biol. 12, 1245–1250 (2002).

    Article  CAS  Google Scholar 

  14. Kiehart, D. P., Galbraith, C. G., Edwards, K. A., Rickoll, W. L. & Montague, R. A. Multiple forces contribute to cell sheet morphogenesis for dorsal closure in Drosophila. J. Cell Biol. 149, 471–490 (2000).

    Article  CAS  Google Scholar 

  15. Solon, J., Kaya-Copur, A., Colombelli, J. & Brunner, D. Pulsed forces timed by a ratchet-like mechanism drive directed tissue movement during dorsal closure. Cell 137, 1331–1342 (2009).

    Article  Google Scholar 

  16. Rodriguez-Diaz, A. et al. Actomyosin purse strings: renewable resources that make morphogenesis robust and resilient. HFSP J. 2, 220–237 (2008).

    Article  Google Scholar 

  17. Hutson, M. S. et al. Forces for morphogenesis investigated with laser microsurgery and quantitative modeling. Science 300, 145–149 (2003).

    Article  CAS  Google Scholar 

  18. Faulkner, G. et al. ZASP: a new Z-band alternatively spliced PDZ-motif protein. J. Cell Biol. 146, 465–475 (1999).

    Article  CAS  Google Scholar 

  19. Zhou, Q. et al. Ablation of Cypher, a PDZ-LIM domain Z-line protein, causes a severe form of congenital myopathy. J. Cell Biol. 155, 605–612 (2001).

    Article  CAS  Google Scholar 

  20. Zhou, Q., Ruiz-Lozano, P., Martone, M. E. & Chen, J. Cypher, a striated muscle-restricted PDZ and LIM domain-containing protein, binds to α-actinin-2 and protein kinase C. J. Biol. Chem. 274, 19807–19813 (1999).

    Article  CAS  Google Scholar 

  21. Jani, K. & Schock, F. Zasp is required for the assembly of functional integrin adhesion sites. J. Cell Biol. 179, 1583–1597 (2007).

    Article  CAS  Google Scholar 

  22. Katzemich, A., Liao, K. A., Czerniecki, S. & Schock, F. Alp/Enigma family proteins cooperate in Z-disc formation and myofibril assembly. PLoS Genet. 9, e1003342 (2013).

    Article  CAS  Google Scholar 

  23. Ducuing, A., Keeley, C., Mollereau, B. & Vincent, S. A DPP-mediated feed-forward loop canalizes morphogenesis during Drosophila dorsal closure. J. Cell Biol. 208, 239–248 (2015).

    Article  CAS  Google Scholar 

  24. Stronach, B. Extensive nonmuscle expression and epithelial apicobasal localization of the Drosophila ALP/Enigma family protein, Zasp52. Gene Expr. Patterns 15, 67–79 (2014).

    Article  CAS  Google Scholar 

  25. Pashmforoush, M. et al. Adult mice deficient in actinin-associated LIM-domain protein reveal a developmental pathway for right ventricular cardiomyopathy. Nat. Med. 7, 591–597 (2001).

    Article  CAS  Google Scholar 

  26. Arimura, T. et al. A Cypher/ZASP mutation associated with dilated cardiomyopathy alters the binding affinity to protein kinase C. J. Biol. Chem. 279, 6746–6752 (2004).

    Article  CAS  Google Scholar 

  27. Selcen, D. & Engel, A. G. Mutations in ZASP define a novel form of muscular dystrophy in humans. Ann. Neurol. 57, 269–276 (2005).

    Article  CAS  Google Scholar 

  28. Vatta, M. et al. Mutations in Cypher/ZASP in patients with dilated cardiomyopathy and left ventricular non-compaction. J. Am. Coll. Cardiol. 42, 2014–2027 (2003).

    Article  CAS  Google Scholar 

  29. Gettings, M. et al. JNK signalling controls remodelling of the segment boundary through cell reprogramming during Drosophila morphogenesis. PLoS Biol. 8, e1000390 (2010).

    Article  Google Scholar 

  30. Katzemich, A., Long, J. Y., Jani, K., Lee, B. R. & Schock, F. Muscle type-specific expression of Zasp52 isoforms in Drosophila. Gene Expr. Patterns 11, 484–490 (2011).

    Article  CAS  Google Scholar 

  31. Gates, J. et al. Enabled plays key roles in embryonic epithelial morphogenesis in Drosophila. Development 134, 2027–2039 (2007).

    Article  CAS  Google Scholar 

  32. Grevengoed, E. E., Loureiro, J. J., Jesse, T. L. & Peifer, M. Abelson kinase regulates epithelial morphogenesis in Drosophila. J. Cell Biol. 155, 1185–1198 (2001).

    Article  CAS  Google Scholar 

  33. Laplante, C. & Nilson, L. A. Differential expression of the adhesion molecule Echinoid drives epithelial morphogenesis in Drosophila. Development 133, 3255–3264 (2006).

    Article  CAS  Google Scholar 

  34. Legoff, L., Rouault, H. & Lecuit, T. A global pattern of mechanical stress polarizes cell divisions and cell shape in the growing Drosophila wing disc. Development 140, 4051–4059 (2013).

    Article  CAS  Google Scholar 

  35. Peralta, X. G. et al. Upregulation of forces and morphogenic asymmetries in dorsal closure during Drosophila development. Biophys. J. 92, 2583–2596 (2007).

    Article  CAS  Google Scholar 

  36. Verma, S. et al. A WAVE2-Arp2/3 actin nucleator apparatus supports junctional tension at the epithelial zonula adherens. Mol. Biol. Cell 23, 4601–4610 (2012).

    Article  CAS  Google Scholar 

  37. Vincent, S., Perrimon, N. & Axelrod, J. D. Hedgehog and Wingless stabilize but do not induce cell fate during Drosophila dorsal embryonic epidermal patterning. Development 135, 2767–2775 (2008).

    Article  CAS  Google Scholar 

  38. Pasakarnis, L., Frei, E., Caussinus, E., Affolter, M. & Brunner, D. Amnioserosa cell constriction but not epidermal actin cable tension autonomously drives dorsal closure. Nat. Cell Biol. http://dx.doi.org/10.1038/ncb3420 (2016).

  39. Caussinus, E., Kanca, O. & Affolter, M. Fluorescent fusion protein knockout mediated by anti-GFP nanobody. Nat. Struct. Mol. Biol. 19, 117–121 (2012).

    Article  CAS  Google Scholar 

  40. Millard, T. H. & Martin, P. Dynamic analysis of filopodial interactions during the zippering phase of Drosophila dorsal closure. Development 135, 621–626 (2008).

    Article  CAS  Google Scholar 

  41. Abate, A. et al. A mathematical model to study the dynamics of epithelial cellular networks. IEEE/ACM Trans. Comput. Biol. Bioinform. 9, 1607–1620 (2012).

    Article  Google Scholar 

  42. Reffay, M. et al. Interplay of RhoA and mechanical forces in collective cell migration driven by leader cells. Nat. Cell Biol. 16, 217–223 (2014).

    Article  CAS  Google Scholar 

  43. Strudwick, X. L. & Cowin, A. J. Cytoskeletal regulation of dermal regeneration. Cells 1, 1313–1327 (2012).

    Article  CAS  Google Scholar 

  44. Li, J., Zhang, S., Soto, X., Woolner, S. & Amaya, E. ERK and phosphoinositide 3-kinase temporally coordinate different modes of actin-based motility during embryonic wound healing. J. Cell Sci. 126, 5005–5017 (2013).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank the ARTHRO-TOOLS and PLATIM facilities of the UMS3444 and Bloomington and the Developmental Studies Hybridoma Bank for reagents. We thank F. Schöck, McGill University, for providing the ZaspΔ mutant, B. Mollereau’s group and M. Grammont’s group for laboratory space and equipment and P. Joncour for running one-way ANOVA and pairwise t-tests in R for Fig. 6f, j. This work was supported by a Chair from the Centre National de la Recherche Scientifique and a COOPERA grant from the Région Rhône-Alpes to S.V. and the Fonds Recherche, ENS de Lyon.

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Authors and Affiliations

  1. LBMC, UMR5239 CNRS/Ecole Normale Supérieure de Lyon, SFR 128 Biosciences Lyon Gerland, Université de Lyon, 69007 Lyon, France

    Antoine Ducuing & Stéphane Vincent

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  1. Antoine Ducuing
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  2. Stéphane Vincent
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Contributions

A.D. performed the experiments, A.D. and S.V. wrote the paper.

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Correspondence to Stéphane Vincent.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 2 Actin cable interruptions correlate with mixer cell formation.

(a) Super-resolution images of WT embryos marked with phalloidin to label actin. Scale bar: 10 μm. Arrowheads indicate interruptions in the actin cable. (b–b′′′) Zasp52::GFP embryo marked with GFP (green in b, grey in b′) and phalloidin to label actin (magenta in b, grey in b′′). (b′′′) Actin intensity of the (b′′) panel color-coded with the ImageJ 16-color LUT. Scale bar: 10 μm. Arrowheads indicate leading edge cells where both Zasp52::GFP and the actin cable are missing. (c–c′′) Zasp52::GFP embryo marked with GFP (blue in c, grey in c′), E-Cadherin (red) and Ena (green in c, grey in c′′). Scale bar: 10 μm. Arrowheads indicate leading edge cells where Zasp52::GFP and the actin cable are missing. Asterisks (∗) indicate the mixer cell that accumulates Ena. Actin cable interruptions correlate with mixer cell formation.

Supplementary Figure 3 Filopodia distribution and Ena accumulation in control and ZaspΔ embryos.

(a-f) Super-resolution images of control (c, d) and ZaspΔ (e-h) embryos marked with phalloidin to label actin (grey). Scale bar: 10 μm. Filopodia are still present in ZaspΔ embryos. (g–i), Prd > Ena (g), Pnr > Ena (h) and ZaspΔ, Pnr > Ena (i) embryos marked with Ena (grey). Scale bar: 10 μm. Ena overexpression in leading edge cells leads to Ena accumulation at the leading edge/amnioserosa interface in a WT (g-h, arrowheads) but not in a ZaspΔ mutant background (i, arrowheads).

Supplementary Figure 4 The actin cable prevents both morphogenetic and wound induced scarring.

(a, b) Still images of Arm::GFP at the end of dorsal closure (a) and ZaspΔ, Arm::GFP (b) after laser surgery in the lateral epidermis. 0 min indicates the closure time. Scale bar: 10 μm. Both morphogenesis and wounding lead to stretches in the epidermis of ZaspΔ embryos. See also Supplementary Video 3 .

Supplementary Figure 5 Ectopic canthi in Puc-lacZ/+ and ZaspΔ; Puc-lacZ/+ embryos.

Puc-lacZ/+ (n = 6) and ZaspΔ; Puc-lacZ/+ (n = 6) embryos marked with E-Cadherin (magenta) and Puc-lacZ (green). Scale bar: 50 μm. Closure is always perfect in Puc-lacZ/+ embryos. In ZaspΔ; Puc-lacZ/+ embryo, the number of ectopic canthi per leading edge is indicated.

Supplementary Figure 6 Folds and stretches in control and ZaspΔ embryos.

(a-b) Control (a) and ZaspΔ (b) embryos marked with E-Cadherin. Scale bar: 10 μm. Both control (n = 6) and ZaspΔ (n = 6) embryos have been acquired with 21 confocal sections separated by a 0.4 μm step. Each panel is a depth color-coding: Z-projection of sections 1 to 7 are in blue, Z-projection of sections 8 to 14 are in red, Z-projection of sections 15 to 21 are in green. In control embryos, the epidermis is flat, and the junctional E-Cadherin staining is detected in the 7 apical-most sections that are in blue. E-Cadherin is also detected in neurons (red) and in tracheas (green). In ZaspΔ embryos, junctional E-Cadherin is detected in intermediate and lower-sections (red and green) and reveal that ectopic canthi produce fold and stretches.

Supplementary Table 1 Statistical reporting.
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Supplementary information

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Zasp52::GFP during dorsal closure.

Live imaging of Zasp52::GFP embryos imaged at 25 °C with a spinning-disk confocal microscope (Leica). GFP is in grey. Frames were acquired every 6 min for 2 h 24. Scale bar: 10 μm. (AVI 652 kb)

Closure dynamic of Control and ZaspΔ embryos.

Live imaging of a Jupiter::GFP/+ and a ZaspΔ, Jupiter::GFP/+ embryo imaged at 25 °C with a spinning-disk confocal microscope (Leica). Jupiter::GFP is in grey. Frames were acquired every 15 min for 6 h. Scale bar: 50 μm. (AVI 769 kb)

Wound healing experiments.

Live imaging of wounded Arm::GFP and ZaspΔ, Arm::GFP embryos imaged at 25 °C with a spinning-disk confocal microscope (Leica). Arm::GFP is in grey. Frames were acquired every 3.5 min for 2 h20. Scale bar: 10 μm. (AVI 3655 kb)

Leading edge straightness of control and ZaspΔ embryos.

Live imaging of a Arm::GFP and ZaspΔ, Arm::GFP embryos imaged at 25 °C with a spinning-disk confocal microscope (Leica). Arm::GFP is in grey. Frames were acquired every 10 min for 4 h30. Scale bar: 25 μm. (AVI 2687 kb)

Single-cell junction cut experiment.

Live imaging of a Arm::GFP embryo imaged at 25 °C with a spinning-disk confocal microscope (Leica) before and after a single-cell junction cut. Arm::GFP is in grey. Frames were acquired every 0.7 s for 2 min. Scale bar: 5 μm. (AVI 520 kb)

Two simultaneous single-cell junction cuts in control and ZaspΔ embryos.

Live imaging of a Arm::GFPand a ZaspΔ, Arm::GFP embryos imaged at 25 °C with a spinning-disk confocal microscope (Leica). Arm::GFP is in grey. Frames were acquired every 5.8 s for 3 min. Scale bar: 10 μm (AVI 2360 kb)

Ectopic canthi formation in ZaspΔ embryos.

Live imaging of a ZaspΔ, Arm::GFP embryo imaged at 25 °C with a spinning-disk confocal microscope (Leica). Arm::GFP is in grey. Frames were acquired every 20 s for 2 h15. Scale bar: 10 μm. (AVI 13984 kb)

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Ducuing, A., Vincent, S. The actin cable is dispensable in directing dorsal closure dynamics but neutralizes mechanical stress to prevent scarring in the Drosophila embryo. Nat Cell Biol 18, 1149–1160 (2016). https://doi.org/10.1038/ncb3421

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