Abstract
Tissue morphogenesis requires coordination of multiple force-producing components. During dorsal closure in fly embryogenesis, an epidermis opening closes. A tensioned epidermal actin/MyosinII cable, which surrounds the opening, produces a force that is thought to combine with another MyosinII force mediating apical constriction of the amnioserosa cells that fill the opening. A model proposing that each force could autonomously drive dorsal closure was recently challenged by a model in which the two forces combine in a ratchet mechanism. Acute force elimination via selective MyosinII depletion in one or the other tissue shows that the amnioserosa tissue autonomously drives dorsal closure while the actin/MyosinII cable cannot. These findings exclude both previous models, although a contribution of the ratchet mechanism at dorsal closure onset remains likely. This shifts the current view of dorsal closure being a combinatorial force-component system to a single tissue-driven closure event.
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References
Sandler, A. D. Children with spina bifida: key clinical issues. Pediatr. Clin. North Am. 57, 879–892 (2010).
Watkins, S. E., Meyer, R. E., Strauss, R. P. & Aylsworth, A. S. Classification, epidemiology, and genetics of orofacial clefts. Clin. Plast. Surg. 41, 149–163 (2014).
Edwards, K. A., Demsky, M., Montague, R. A., Weymouth, N. & Kiehart, D. P. GFP-moesin illuminates actin cytoskeleton dynamics in living tissue and demonstrates cell shape changes during morphogenesis in Drosophila. Dev. Biol. 191, 103–117 (1997).
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).
Franke, J. D., Montague, R. A., Kiehart, D. P. & Carolina, N. Nonmuscle myosin II generates forces that transmit tension and drive contraction in multiple tissues during dorsal closure. Curr. Biol. 15, 2208–2221 (2005).
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).
Eltsov, M. et al. Quantitative analysis of cytoskeletal reorganization during epithelial tissue sealing by large-volume electron tomography. Nat. Cell Biol. 17, 605–614 (2015).
Hutson, M. S. et al. Forces for morphogenesis investigated with laser microsurgery and quantitative modeling. Science 300, 145–149 (2003).
Peralta, X. G. et al. Upregulation of forces and morphogenic asymmetries in dorsal closure during Drosophila development. Biophys. J. 92, 2583–2596 (2007).
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).
David, D. J. V., Tishkina, A. & Harris, T. J. C. The PAR complex regulates pulsed actomyosin contractions during amnioserosa apical constriction in Drosophila. Development 137, 1645–1655 (2010).
Blanchard, G. B., Murugesu, S., Adams, R. J., Martinez-Arias, A. & Gorfinkiel, N. Cytoskeletal dynamics and supracellular organisation of cell shape fluctuations during dorsal closure. Development 137, 2743–2752 (2010).
Saravanan, S., Meghana, C. & Narasimha, M. Local, cell-nonautonomous feedback regulation of myosin dynamics patterns transitions in cell behavior: a role for tension and geometry? Mol. Biol. Cell 24, 2350–2361 (2013).
Jacinto, A. et al. Dynamic analysis of actin cable function during Drosophila dorsal closure. Curr. Biol. 12, 1245–1250 (2002).
Scuderi, A. & Letsou, A. Amnioserosa is required for dorsal closure in Drosophila. Dev. Dyn. 232, 791–800 (2005).
Wada, A., Kato, K., Uwo, M. F., Yonemura, S. & Hayashi, S. Specialized extraembryonic cells connect embryonic and extraembryonic epidermis in response to Dpp during dorsal closure in Drosophila. Dev. Biol. 301, 340–349 (2007).
Wells, A. R. et al. Complete canthi removal reveals that forces from the amnioserosa alone are sufficient to drive dorsal closure in Drosophila. Mol. Biol. Cell 25, 3552–3568 (2014).
Rodriguez-Diaz, A. et al. Actomyosin purse strings: renewable resources that make morphogenesis robust and resilient. HFSP J. 2, 220–237 (2008).
Caussinus, E., Kanca, O. & Affolter, M. Fluorescent fusion protein knockout mediated by anti-GFP nanobody. Nat. Struct. Mol. Biol. 19, 117–121 (2012).
Mizuno, T., Tsutsui, K. & Nishida, Y. Drosophila myosin phosphatase and its role in dorsal closure. Development 129, 1215–1223 (2002).
Wodarz, A., Hinz, U., Engelbert, M. & Knust, E. Expression of crumbs confers apical character on plasma membrane domains of ectodermal epithelia of Drosophila. Cell 82, 67–76 (1995).
Manseau, L. et al. GAL4 enhancer traps expressed in the embryo, larval brain, imaginal discs, and ovary of Drosophila. Dev. Dyn. 209, 310–322 (1997).
Chatterjee, N. & Bohmann, D. A versatile φC31 based reporter system for measuring AP-1 and NRF2 signaling in Drosophila and in tissue culture. PLoS One 7, e34063 (2012).
Fischer, S. C. et al. Contractile and mechanical properties of epithelia with perturbed actomyosin dynamics. PLoS One 9, e95695 (2014).
Lee A, T. J. Excessive Myosin activity in mbs mutants causes photoreceptor movement out of the Drosophila eye disc epithelium. Mol. Biol. Cell 15, 3285–3295 (2004).
Saias, L. et al. Decrease in cell volume generates contractile forces driving dorsal closure. Dev. Cell 33, 611–621 (2015).
Azevedo, D. et al. DRhoGEF2 regulates cellular tension and cell pulsations in the amnioserosa during Drosophila dorsal closure. PLoS One 6, e23964 (2011).
Ma, X., Lynch, H. E., Scully, P. C. & Hutson, M. S. Probing embryonic tissue mechanics with laser hole drilling. Phys. Biol. 6, 036004 (2009).
Gorfinkiel, N., Blanchard, G. B., Adams, R. J. & Martinez Arias, A. Mechanical control of global cell behaviour during dorsal closure in Drosophila. Development 136, 1889–1898 (2009).
Herranz, H. & Morata, G. The functions of pannier during Drosophila embryogenesis. Development 128, 4837–4846 (2001).
Brand, A. H. & Perrimon, N. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118, 401–415 (1993).
Glise, B. & Noselli, S. Coupling of Jun amino-terminal kinase and Decapentaplegic signaling pathways in Drosophila morphogenesis. Genes Dev. 11, 1738–1747 (1997).
Pfeiffer, B. D. et al. Tools for neuroanatomy and neurogenetics in Drosophila. Proc. Natl Acad. Sci. USA 105, 9715–9720 (2008).
Takaesu, N. T., Johnson, A. N. & Newfeld, S. J. Posterior spiracle specific GAL4 lines: new reagents for developmental biology and respiratory physiology. Genesis 34, 16–18 (2002).
Sepp, K. J. & Auld, V. J. Conversion of lacZ enhancer trap lines to GAL4 lines using targeted transposition in Drosophila melanogaster. Genetics 151, 1093–1101 (1999).
Martín-Blanco, E. et al. Puckered encodes a phosphatase that mediates a feedback loop regulating JNK activity during dorsal closure in Drosophila. Genes Dev. 12, 557–670 (1998).
Jankovics, F. & Brunner, D. Transiently reorganized microtubules are essential for zippering during dorsal closure in Drosophila melanogaster. Dev. Cell 11, 375–385 (2006).
David, D. J. V., Wang, Q., Feng, J. J. & Harris, T. J. C. Bazooka inhibits aPKC to limit antagonism of actomyosin networks during amnioserosa apical constriction. Development 140, 4719–4729 (2013).
Sokolow, A., Toyama, Y., Kiehart, D. P. & Edwards, G. S. Cell ingression and apical shape oscillations during dorsal closure in Drosophila. Biophys. J. 102, 969–979 (2012).
Jayasinghe, A. K., Crews, S. M., Mashburn, D. N. & Hutson, M. S. Apical oscillations in amnioserosa cells: basolateral coupling and mechanical autonomy. Biophys. J. 105, 255–265 (2013).
Narasimha, M. & Brown, N. H. Novel functions for integrins in epithelial morphogenesis. Curr. Biol. 14, 381–385 (2004).
Machado, P. F. et al. Emergent material properties of developing epithelial tissues. BMC Biol. 13, 98 (2015).
Lehmann, R. & Nüsslein-Volhard, C. Abdominal segmentation, pole cell formation, and embryonic polarity require the localized activity of oskar, a maternal gene in Drosophila. Cell 47, 141–152 (1986).
Franke, J. D., Montague, R. A. & Kiehart, D. P. Nonmuscle myosin II is required for cell proliferation, cell sheet adhesion and wing hair morphology during wing morphogenesis. Dev. Biol. 345, 117–132 (2010).
Wang, Q., Feng, J. J. & Pismen, L. M. A cell-level biomechanical model of Drosophila dorsal closure. Biophys. J. 103, 2265–2274 (2012).
Martin, A. C., Kaschube, M. & Wieschaus, E. F. Pulsed contractions of an actin–myosin network drive apical constriction. Nature 457, 495–499 (2009).
Rauzi, M., Lenne, P.-F. & Lecuit, T. Planar polarized actomyosin contractile flows control epithelial junction remodelling. Nature 468, 1110–1114 (2010).
Gorfinkiel, N. & Arias, A. M. Requirements for adherens junction components in the interaction between epithelial tissues during dorsal closure in Drosophila. J. Cell Sci. 120, 3289–3298 (2007).
Kaltschmidt, J. A. et al. Planar polarity and actin dynamics in the epidermis of Drosophila. Nat. Cell Biol. 4, 937–944 (2002).
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. http://dx.doi.org/10.1038/ncb3421 (2016).
Igaki, T. et al. Drob-1, a Drosophila member of the Bcl-2/CED-9 family that promotes cell death. Proc. Natl Acad. Sci. USA 97, 662–667 (2000).
Rubin, G. M. & Spradling, A. C. Genetic transformation of Drosophila with transposable element vectors. Science 218, 348–353 (1982).
Abreu-Blanco, M. T., Verboon, J. M. & Parkhurst, S. M. Cell wound repair in Drosophila occurs through three distinct phases of membrane and cytoskeletal remodeling. J. Cell Biol. 193, 455–464 (2011).
Acknowledgements
We thank C. Lehner and M. Daellenbach for critical reading of the manuscript. We are grateful to W. Boll for providing help with the microscopy. We thank A. Trenner and W. Boll for support in generating mCherry-Zipper. We also thank the many people providing fly strains. L.P. was supported in part by a grant from the University of Zurich and, together with E.C., by the Swiss initiative in Systems Biology, SystemsX.ch (MorphogenetiX). M.A. acknowledges funding from the Cantons Basel-Stadt and Basel-Land and from the SNSF. D.B. acknowledges financial support from the University of Zurich.
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L.P. and D.B. designed the experiments; L.P. performed the experiments; L.P. and E.F. generated fly strains; E.C. and M.A. provided and shared unpublished information about the development and use of the deGradFP system; L.P. and D.B. analysed the data and wrote the manuscript. All authors proofread the manuscript.
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Supplementary Figure 1 Selective Sqh-GFP depletion in amnioserosa cells.
(a) UAS-mCherry-moesin expression pattern of the most commonly used amnioserosa-specific Gal4 enhancer elements 332.3Gal4 and c381Gal4. Arm-GFP outlines the amnioserosa tissue in the embryos with leaky c381Gal4 expression. Scale bar: 50 μm. (b) Selected movie frames showing dorsal closure progression in AS-SqhKO embryos, expressing rescuing Sqh-mCherry. Left column shows Sqh-GFP signal, right column show Sqh-mCherry signal. Scale bar: 50 μm. Images in a and b are representative of >10 embryos from >2 experiments. (c) Sqh-GFP-expressing embryos, selectively expressing UAS-Mbs.N300 in the amnioserosa tissue. Scale bar: 20 μm. Image represents one experiment with 4 embryos. (d) Relative gap area change (%/min) in control (black, n = 19 embryos, pooled from 3 independent experiments) and AS-SqhKO embryos (red, n = 23 embryos, pooled from 4 independent experiments). Mann-Whitney U test: ∗P < 0.05, ∗∗∗P < 0.001. Dashed lines: Standard deviation. (e) Convergence speeds of the two lateral LEs in control (black, 19 embryos, pooled from 3 independent experiments) and AS-SqhKO embryos (red, 23 embryos, pooled from 4 independent experiments). Dashed lines: Standard deviation. (f) Gap length changes in control (black, n = 19 embryos, pooled from 3 independent experiments) and AS-SqhKO embryos (red, n = 23 embryos, pooled from 4 independent experiments). Mann-Whitney U test: ∗P < 0.05, ∗∗∗P < 0.001. Dashed lines: Standard deviation. Statistics source data for d and f is provided in Supplementary Table 2.
Supplementary Figure 2 Sqh-GFP depletion in epidermis cells with pnr enhancer element.
(a) Selected movie frames showing germ band retraction and dorsal closure failure in pnrES-SqhKO embryos, with the leaky pnr enhancer element. The dashed red line represents the LE. Cartoon: In green are cells with functional Sqh-GFP. Scale bar: 50 μm. (b) 3 examples of embryos expressing deGradFP with the leaky pnr enhancer element. One example each of an affected, expanding amnioserosa cell and an unaffected, constricting amnioserosa cell is outlined in red and blue respectively. Scale bar: 20 μm. (c) Schematic showing strategy for Gal4 swapping with Gal80 in the 332.3 locus. (d) Embryos in which the leaky pnr enhancer element was used to drive expression of the actin-marking mCherry-moesin without (top) and in combination with 332.3Gal80 (bottom). Red arrows indicate amnioserosa cells with ectopic mCherry-moesin expression by pnr. Scale bar: 10 μm. Images in a,b and d are representative of >10 embryos from >3 experiments.
Supplementary Figure 3 Phenotypes of dorsal closure in ES-SqhKO embryos.
(a) mCherry-moesin (labels F-actin) expressing control and ES-SqhKO embryos at the end of dorsal closure. 8% of the embryos show no puckering defects (n = 89), 65% show mild puckering defects (red arrows) and 27% show a severe puckering defect. Scale bar: 50 μm. Images are representative of 89 embryos from >5 experiments. (b) mCherry-moesin (labels F-actin) expressing control and ES-SqhKO embryos at the end of dorsal closure. In 41% of the embryos (n = 95) the anterior epidermis ruptures. Scale bar: 50 μm. Images are representative of 95 embryos from >5 experiments. (c) Recoil of epidermis (Sqh-GFP labelled) after laser incision in control and ES-SqhKO embryos. Red arrows indicate the extent of tissue recoil, black arrows show the direction of the laser cut. Scale bar: 20 μm. Images are representative of 4 control and 3 ES-SqhKO laser incisions/embryos from 1 experiment. (d) Gap width change in control (black, n = 19 embryos, pooled from 3 independent experiments) and ES-SqhKO embryos (red, n = 26 embryos, pooled from 6 independent experiments). Dashed lines: Standard deviation. Mann-Whitney U test: ∗P < 0.05. (e) Convergence speeds of the two lateral LEs in control (black, 19 embryos, pooled from 3 independent experiments) and ES-SqhKO embryos (red, 26 embryos, pooled from 6 independent experiments). The right panel is an overlay of the first two panels. Statistics source data for d and e is provided in Supplementary Table 2.
Supplementary Figure 4 F-actin dynamics/tissue tension in AS-SqhKO and ES-SqhKO embryos.
(a) Comparison of Sqh-GFP intensity oscillations over time in amnioserosa cells of control and AS-SqhKO embryos. For the control 3 of 12 analysed cells from 2 embryos in 2 independent experiments are shown. For AS-SqhKO 3 of 24 analysed cells from 5 embryos in 4 independent experiments are shown. Statistics source data is provided in Supplementary Table 2. (b) Comparison of F-actin in amnioserosa cells of control and AS-SqhKO embryos. Scale bar: 10 μm. Images are representative of 2 control and 5 AS-SqhKO embryos from 2 experiments. (c) Comparison of the apical amnioserosa cell surface area dynamics in control (black/grey) and AS-SqhKO (red/orange) embryos. For the control 8 of 16 analysed cells from 2 embryos in 2 independent experiments are shown. For AS-SqhKO 8 of 29 analysed cells from 4 embryos in 3 independent experiments are shown. Statistics source data is provided in Supplementary Table 2. (d) Sqh-GFP degradation aggregates and F-actin localization (mCherry-moesin labelled) in AS-SqhKO embryos. Scale bar: 20 μm. Images are representative of >10 embryos from >5 experiments. (e) Vertex recoil in the first 12s following a laser incision of a junction connecting 2 amnioserosa cells in control (black, 157 vertices, pooled from 22 embryos in 8 independent experiments) and AS-SqhKO embryos (red, 104 vertices, pooled from 17 embryos in 4 independent experiments). Displacement values of all tracked cell vertices were pooled for each time point. Error bars: Standard deviation. Statistics source data is provided in Supplementary Table 2. (f) Illustration of the method to set the angle between a tracked cell vertex and the ablated junction. The two vertices delimiting an ablated junction have an angle of 0° (orange line). All other vertices are at a certain angle from the ablated junction (red line, max. ±90°). The star indicates the incision point. Scale bar: 10 μm. Image is representative of 22 control and 17 AS-SqhKO embryos from >5 experiments. (g) Procedure for LE intensity measurements (Methods). The yellow rectangle indicates the 80 30-pixel lines (red) and the way they were placed on the LE. The 3rd frame shows an example of one set of 80 average intensity values obtained from measurements in one yellow rectangle. All measurements for each of the 80 values were assembled into box plots in Fig. 3c. In green: Position of the 20 30-pixel lines the average of which was used to normalise the intensity profile. Scale bar: 10 μm. Images are representative of 7 control, 11 ES-SqhKO, and 9 Mbs.N300 embryos from 7 independent experiments in total.
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Germ-band retraction and dorsal closure progression of a control and an AS-SqhKO embryo.
In addition to dorsal closure, movies at this resolution reveal midgut closure, which occurs below the amnioserosa tissue towards the end of dorsal closure (left halve of amnioserosa tissue in the wild type, middle of the amnioserosa tissue in AS-SqhKO), and which is not affected by dorsal closure inhibition. Time point 0 min marks the end of germ-band retraction in all movies. Scale bar: 50 μm. (MOV 3962 kb)
Germ-band retraction and dorsal closure progression of a control and a pnrES-SqhKO embryo in which some amnioserosa cells express deGradFP due to pnr leakiness.
Scale bar: 50 μm. (MOV 1779 kb)
Germ-band retraction and dorsal closure progression of a control and a ES-SqhKO embryo in which ‘leaky’ pnrGal4 expression was suppressed in the amnioserosa-tissue with 332.3Gal80.
Scale bar: 50 μm. (MOV 3895 kb)
Zipping LEs of an ES-SqhKO embryo.
Epidermis cells express mCherry-moesin to label F-actin. Scale bar: 20 μm. (MOV 3771 kb)
Zipping LEs of an enES-SqhKO embryo.
Engrailed expressing cells express mCherry-moesin to label F-actin. Scale bar: 20 μm. (MOV 3473 kb)
Gradual apical and junctional Sqh-GFP accumulation in amnioserosa cells of control and ES-SqhKO embryos.
Sequential, junctional Sqh-GFP accumulation becomes visible at around 100 min while apical Sqh.GFP accumulation in peripheral amnioserosa cells becomes evident at 200 min. Scale bar: 20 μm. (MOV 2984 kb)
Sqh-GFP in amnioserosa cells of control and AS-SqhKO embryos.
Scale bar: 20 μm. (MOV 1719 kb)
Laser incision of amnioserosa cell–cell junctions in control and AS-SqhKO embryos.
Scale bar: 20 μm. (MOV 3392 kb)
F-actin intensity measurements at the LE of control, ES-SqhKO and ES-Mbs.N300 embryos.
Shown is the placement of the 30 × 80 pixel rectangles. Scale bar: 20 μm. (MOV 1879 kb)
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Pasakarnis, L., Frei, E., Caussinus, E. et al. Amnioserosa cell constriction but not epidermal actin cable tension autonomously drives dorsal closure. Nat Cell Biol 18, 1161–1172 (2016). https://doi.org/10.1038/ncb3420
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DOI: https://doi.org/10.1038/ncb3420
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