Abstract
Gastrulation is the first major morphogenetic event during ascidian embryogenesis. Ascidian gastrulation begins with the invagination of the endodermal progenitors, a two-step process driven by individual cell shape changes of endoderm cells. During the first step, endoderm cells constrict apically, thereby flattening the vegetal side of the embryo. During the second step, endoderm cells shorten along their apicobasal axis and tissue invagination ensues. Individual cell shape changes are mediated by localized actomyosin contractile activity. Here, we describe methods used during ascidian endoderm apical constriction to study myosin activity and cellular morphodynamics with confocal and light sheet microscopy and followed by quantitative image analysis.
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References
Guignard L et al (2020) Contact area-dependent cell communication and the morphological invariance of ascidian embryogenesis. Science 369(6500):eaar5663. https://doi.org/10.1126/science.aar5663
Leggio B, Laussu J, Carlier A, Godin C, Lemaire P, Faure E (2019) MorphoNet: an interactive online morphological browser to explore complex multi-scale data. Nat Commun 10(1):2812. https://doi.org/10.1038/s41467-019-10668-1
Sherrard K, Robin F, Lemaire P, Munro E (2010) Sequential activation of apical and basolateral contractility drives ascidian endoderm invagination. Curr Biol 20(17):1499–1510. https://doi.org/10.1016/j.cub.2010.06.075
Fiuza U-M, Negishi T, Rouan A, Yasuo H, Lemaire P (2020) A Nodal/Eph signalling relay drives the transition from apical constriction to apico-basal shortening in ascidian endoderm invagination. Development 147(15):dev186965. https://doi.org/10.1242/dev.186965
Hashimoto H, Robin FB, Sherrard KM, Munro EM (2015) Sequential contraction and exchange of apical junctions drives zippering and neural tube closure in a simple chordate. Dev Cell 32(2):241–255. https://doi.org/10.1016/j.devcel.2014.12.017
Weber M, Huisken J (2011) Light sheet microscopy for real-time developmental biology. Curr Opin Genet Dev 21(5):566–572. https://doi.org/10.1016/j.gde.2011.09.009
Siedentopf H, Zsigmondy R (1902) Uber Sichtbarmachung und Größenbestimmung ultramikoskopischer Teilchen, mit besonderer Anwendung auf Goldrubingläser. Ann Phys 315(1):1–39. https://doi.org/10.1002/andp.19023150102
Voie AH, Burns DH, Spelman FA (1993) Orthogonal-plane fluorescence optical sectioning: three-dimensional imaging of macroscopic biological specimens. J Microsc 170(3):229–236. https://doi.org/10.1111/j.1365-2818.1993.tb03346.x
Huisken J, Swoger J, Del Bene F, Wittbrodt J, Stelzer EHK (2004) Optical sectioning deep inside live embryos by selective plane illumination microscopy. Science 305(5686):1007–1009. https://doi.org/10.1126/science.1100035
Keller PJ, Stelzer EHK (2008) Quantitative in vivo imaging of entire embryos with digital scanned laser light sheet fluorescence microscopy. Curr Opin Neurobiol 18(6):624–632. https://doi.org/10.1016/j.conb.2009.03.008
Daetwyler S, Huisken J (2016) Fast fluorescence microscopy with light sheets. Biol Bull 231(1):14–25. https://doi.org/10.1086/689588
Huisken J, Stainier DYR (2007) Even fluorescence excitation by multidirectional selective plane illumination microscopy (mSPIM). Opt Lett 32(17):2608–2610. https://doi.org/10.1364/ol.32.002608
Krzic U, Gunther S, Saunders TE, Streichan SJ, Hufnagel L (2012) Multiview light-sheet microscope for rapid in toto imaging. Nat Methods 9(7):730–733. https://doi.org/10.1038/nmeth.2064
Tomer R, Khairy K, Amat F, Keller PJ (2012) Quantitative high-speed imaging of entire developing embryos with simultaneous multiview light-sheet microscopy. Nat Methods 9(7):755–763. https://doi.org/10.1038/nmeth.2062
Chhetri RK, Amat F, Wan Y, Höckendorf B, Lemon WC, Keller PJ (2015) Whole-animal functional and developmental imaging with isotropic spatial resolution. Nat Methods 12(12):1171–1178. https://doi.org/10.1038/nmeth.3632
Wu Y et al (2011) Inverted selective plane illumination microscopy (iSPIM) enables coupled cell identity lineaging and neurodevelopmental imaging in Caenorhabditis elegans. Proc Natl Acad Sci 108(43):17708–17713. https://doi.org/10.1073/pnas.1108494108
Kumar A et al (2014) Dual-view plane illumination microscopy for rapid and spatially isotropic imaging. Nat Protoc 9(11):2555–2573. https://doi.org/10.1038/nprot.2014.172
Strnad P et al (2016) Inverted light-sheet microscope for imaging mouse pre-implantation development. Nat Methods 13(2):139–142. https://doi.org/10.1038/nmeth.3690
de Medeiros G, Norlin N et al (2015) Confocal multiview light-sheet microscopy. Nat Commun 6:8881. https://doi.org/10.1038/ncomms9881
Prodon F et al (2010) Dual mechanism controls asymmetric spindle position in ascidian germ cell precursors. Development 137(12):2011–2021. https://doi.org/10.1242/dev.047845
Hotta K et al (2007) A web-based interactive developmental table for the ascidian Ciona intestinalis, including 3D real-image embryo reconstructions: I. From fertilized egg to hatching larva. Dev Dyn 236(7):1790–1805. https://doi.org/10.1002/dvdy.21188
Carroll M, Levasseur M, Wood C, Whitaker M, Jones KT, McDougall A (2003) Exploring the mechanism of action of the sperm-triggered calcium-wave pacemaker in ascidian zygotes. J Cell Sci 116(24):4997–5004. https://doi.org/10.1242/jcs.00846
Stegmaier J et al (2015) Real-time three-dimensional cell segmentation in large-scale microscopy data of developing embryos. Dev Cell 36(2):225–240. https://doi.org/10.1016/j.devcel.2015.12.028
Melak M, Plessner M, Grosse R (2017) Actin visualization at a glance. J Cell Sci 130(3):525–530. https://doi.org/10.1242/jcs.189068
Passamaneck YJ, Hadjantonakis A-K, Di Gregorio A (2007) Dynamic and polarized muscle cell behaviors accompany tail morphogenesis in the ascidian Ciona intestinalis. PLoS One 2(8):e714. https://doi.org/10.1371/journal.pone.0000714
Prodon F, Hanawa K, Nishida H (2009) Actin microfilaments guide the polarized transport of nuclear pore complexes and the cytoplasmic dispersal of Vasa mRNA during GVBD in the ascidian Halocynthia roretzi. Dev Biol 330(2):377–388. https://doi.org/10.1016/j.ydbio.2009.04.006
Sensui N, Yoshida M, Morisawa M (2001) Roles of MLCK and PI3 kinase on deformation and ooplasmic segregation at fertilization in the egg of Ciona savignyi. In: Sawada H, Yokosawa H, Lambert CC (eds) The biology of Ascidians. Springer, Tokyo, pp 92–96
Guglielmi G, Falk HJ, De Renzis S (2016) Optogenetic control of protein function: from intracellular processes to tissue morphogenesis. Trends Cell Biol 26(11):864–874. https://doi.org/10.1016/j.tcb.2016.09.006
Krueger D, Izquierdo E, Viswanathan R, Hartmann J, Pallares Cartes C, De Renzis S (2019) Principles and applications of optogenetics in developmental biology. Development 146(20):22. https://doi.org/10.1242/dev.175067
Colombelli J, Solon J (2013) Force communication in multicellular tissues addressed by laser nanosurgery. Cell Tissue Res 352(1):133–147. https://doi.org/10.1007/s00441-012-1445-1
Schindelin J et al (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9(7):676–682. https://doi.org/10.1038/nmeth.2019
Preibisch S, Saalfeld S, Schindelin J, Tomancak P (2010) Software for bead-based registration of selective plane illumination microscopy data. Nat Methods 7(6):418–419. https://doi.org/10.1038/nmeth0610-418
Weigert M et al (2018) Content-aware image restoration: pushing the limits of fluorescence microscopy. Nat Methods 15(12):1090–1097. https://doi.org/10.1038/s41592-018-0216-7
Berg S et al (2019) ilastik: interactive machine learning for (bio)image analysis. Nat Methods 16(12):1226–1232. https://doi.org/10.1038/s41592-019-0582-9
Fernandez R et al (2010) Imaging plant growth in 4D: robust tissue reconstruction and lineaging at cell resolution. Nat Methods 7(7):547–553. https://doi.org/10.1038/nmeth.1472
Mosaliganti KR, Noche RR, Xiong F, Swinburne IA, Megason SG (2012) ACME: automated cell morphology extractor for comprehensive reconstruction of cell membranes. PLoS Comput Biol 8(12):e1002780. https://doi.org/10.1371/journal.pcbi.1002780
Wolff C et al (2018) Multi-view light-sheet imaging and tracking with the MaMuT software reveals the cell lineage of a direct developing arthropod limb. eLife 7:e34410. https://doi.org/10.7554/eLife.34410
Arganda-Carreras I et al (2020) ijpb/MorphoLibJ: MorphoLibJ 1.4.2.1. Zenodo, 2020
Caputi L, Andreakis N, Mastrototaro F, Cirino P, Vassillo M, Sordino P (2007) Cryptic speciation in a model invertebrate chordate. Proc Natl Acad Sci 104(22):9364–9369. https://doi.org/10.1073/pnas.0610158104
Brunetti R, Gissi C, Pennati R, Caicci F, Gasparini F, Manni L (2015) Morphological evidence that the molecularly determined Ciona intestinalis type A and type B are different species: Ciona robusta and Ciona intestinalis. J Zool Syst Evol Res 53(3):186–193. https://doi.org/10.1111/jzs.12101
Okado H, Takahashi K (1993) Neural differentiation in cleavage-arrested ascidian blastomeres induced by a proteolytic enzyme. J Physiol 463:269–290. https://doi.org/10.1113/jphysiol.1993.sp019594
Acknowledgments
This work was supported by the CNRS and the Agence Nationale de la Recherche (Dig-Em project, ANR-14-CE11-0013-01; Morphoscope2 Equipex, ANR-11-EQPX-0029). U.M.F. was supported by the Dig-Em project, by the FRM (SPF20120523969) and by the EMBL Interdisciplinary Postdoc Programme under Marie Curie Actions. P.L. is a CNRS senior staff scientist. We thank Carla Pérez (currently at EPFL, Lausanne) for sharing her Master internship results optimizing Phallusia mammillata salinity incubation conditions. We thank B. Balázs and O. Selchow for discussions.
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Fiúza, UM., Lemaire, P. (2022). Methods for the Study of Apical Constriction During Ascidian Gastrulation. In: Chang, C., Wang, J. (eds) Cell Polarity Signaling. Methods in Molecular Biology, vol 2438. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2035-9_23
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DOI: https://doi.org/10.1007/978-1-0716-2035-9_23
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