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Vertebrate Embryogenesis pp 17–32Cite as

Immunofluorescence of Microtubule Assemblies in Amphibian Oocytes and Early Embryos

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1920))

Abstract

Amphibian oocytes and embryos are classical models to study cellular and developmental processes. For these studies, it is often advantageous to visualize protein organization. However, the large size and yolk distribution make imaging of deep structures in amphibian zygotes challenging. Here we describe in detail immunofluorescence (IF) protocols for imaging microtubule assemblies in early amphibian development. We developed these protocols to elucidate how the cell division machinery adapts to drastic changes in embryonic cell sizes. We describe how to image mitotic spindles, microtubule asters, chromosomes, and nuclei in whole-mount embryos, even when they are hundreds of micrometers removed from the embryo’s surface. Though the described methods were optimized for microtubule assemblies, they have also proven useful for the visualization of other proteins.

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References

  1. Swammerdam J (1737) Bibilia Naturae; sive historia insectorum, in classes certas redact 2

    Google Scholar 

  2. Baker JR (1951) Remarks on the discovery of cell-division. Isis 42(130):285–287

    Article  CAS  PubMed  Google Scholar 

  3. Hertwig O (1893) Ueber den Werth der ersten Furchungszellen fuer die Organbildung des Embryo. Experimentelle Studien am Frosch- und Tritonei. Arch mikr Anat xlii:662–807

    Article  Google Scholar 

  4. Pflüger E (1884) Ueber die Einwirkung der Schwerkraft und anderer Bedingungen auf die Richtung der Zelltheilung. Pflügers Arch Eur J Physiol 34(1):607–616. https://doi.org/10.1007/BF01612880

    Article  Google Scholar 

  5. Wallingford JB, Liu KJ, Zheng Y (2010) Xenopus. Curr Biol 20(6):R263–R264. https://doi.org/10.1016/j.cub.2010.01.012

    Article  CAS  PubMed  Google Scholar 

  6. Lohka MJ, Maller JL (1985) Induction of nuclear envelope breakdown, chromosome condensation, and spindle formation in cell-free extracts. J Cell Biol 101(2):518–523

    Article  CAS  PubMed  Google Scholar 

  7. Sawin KE, Mitchison TJ (1991) Mitotic spindle assembly by two different pathways in vitro. J Cell Biol 112(5):925–940

    Article  CAS  PubMed  Google Scholar 

  8. Reinsch S, Karsenti E (1997) Movement of nuclei along microtubules in Xenopus egg extracts. Curr Biol 7(3):211–214

    Article  CAS  PubMed  Google Scholar 

  9. Lohka MJ, Masui Y (1983) Formation in vitro of sperm pronuclei and mitotic chromosomes induced by amphibian ooplasmic components. Science 220(4598):719–721

    Article  CAS  PubMed  Google Scholar 

  10. Coleman TR, Carpenter PB, Dunphy WG (1996) The Xenopus Cdc6 protein is essential for the initiation of a single round of DNA replication in cell-free extracts. Cell 87(1):53–63

    Article  CAS  PubMed  Google Scholar 

  11. Levy DL, Heald R (2010) Nuclear size is regulated by importin alpha and Ntf2 in Xenopus. Cell 143(2):288–298. https://doi.org/10.1016/j.cell.2010.09.012

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Murray AW (1991) Cell cycle extracts. Methods Cell Biol 36:581–605

    Article  CAS  PubMed  Google Scholar 

  13. Wühr M, Dumont S, Groen AC, Needleman DJ, Mitchison TJ (2009) How does a millimeter-sized cell find its center. Cell Cycle 8(8):1115–1121

    Article  PubMed  Google Scholar 

  14. Wühr M, Chen Y, Dumont S, Groen AC, Needleman DJ, Salic A, Mitchison TJ (2008) Evidence for an upper limit to mitotic spindle length. Curr Biol 18(16):1256–1261. https://doi.org/10.1016/j.cub.2008.07.092

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Mitchison T, Wühr M, Nguyen P, Ishihara K, Groen A, Field CM (2012) Growth, interaction, and positioning of microtubule asters in extremely large vertebrate embryo cells. Cytoskeleton (Hoboken) 69(10):738–750. https://doi.org/10.1002/cm.21050

    Article  CAS  Google Scholar 

  16. Field CM, Pelletier JF, Mitchison TJ (2017) Xenopus extract approaches to studying microtubule organization and signaling in cytokinesis. Methods Cell Biol 137:395–435. https://doi.org/10.1016/bs.mcb.2016.04.014

    Article  CAS  PubMed  Google Scholar 

  17. Wühr M, Güttler T, Peshkin L, McAlister GC, Sonnett M, Ishihara K, Groen AC, Presler M, Erickson BK, Mitchison TJ, Kirschner MW, Gygi SP (2015) The nuclear proteome of a vertebrate. Curr Biol 25(20):2663–2671. https://doi.org/10.1016/j.cub.2015.08.047

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Peshkin L, Wühr M, Pearl E, Haas W, Freeman RM Jr, Gerhart JC, Klein AM, Horb M, Gygi SP, Kirschner MW (2015) On the relationship of protein and mRNA dynamics in vertebrate embryonic development. Dev Cell 35(3):383–394. https://doi.org/10.1016/j.devcel.2015.10.010

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Lombard-Banek C, Moody SA, Nemes P (2016) High-sensitivity mass spectrometry for probing gene translation in single embryonic cells in the early frog (Xenopus) embryo. Front Cell Dev Biol 4:100. https://doi.org/10.3389/fcell.2016.00100

    Article  PubMed  PubMed Central  Google Scholar 

  20. Smits AH, Lindeboom RG, Perino M, van Heeringen SJ, Veenstra GJ, Vermeulen M (2014) Global absolute quantification reveals tight regulation of protein expression in single Xenopus eggs. Nucleic Acids Res 42(15):9880–9891. https://doi.org/10.1093/nar/gku661

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Gurdon JB, Wickens MP (1983) The use of Xenopus oocytes for the expression of cloned genes. Methods Enzymol 101:370–386

    Article  CAS  PubMed  Google Scholar 

  22. Schultze O (1887) Untersuchungen uber die Reifung und Befruchtung des Amphibieneies. Erste Abhandlung. Z wiss Zool xlv:177–226

    Google Scholar 

  23. Kieserman EK, Lee C, Gray RS, Park TJ, Wallingford JB (2010) High-magnification in vivo imaging of Xenopus embryos for cell and developmental biology. Cold Spring Harb Protoc 2010(5):pdb prot5427. https://doi.org/10.1101/pdb.prot5427

    Article  PubMed  Google Scholar 

  24. Karasaki S (1963) Studies on amphibian yolk 1. The ultrastructure of the yolk platelet. J Cell Biol 18:135–151

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Karasaki S (1963) Studies on amphibian yolk. 5. Electron microscopic observations on the utilization of yolk platelets during embryogenesis. J Ultrastruct Res 59:225–247

    Article  CAS  PubMed  Google Scholar 

  26. Dent JA, Polson AG, Klymkowsky MW (1989) A whole-mount immunocytochemical analysis of the expression of the intermediate filament protein vimentin in Xenopus. Development 105(1):61–74

    CAS  PubMed  Google Scholar 

  27. Becker BE, Gard DL (2006) Visualization of the cytoskeleton in Xenopus oocytes and eggs by confocal immunofluorescence microscopy. Methods Mol Biol 322:69–86

    Article  PubMed  Google Scholar 

  28. Wühr M, Tan ES, Parker SK, Detrich HW 3rd, Mitchison TJ (2010) A model for cleavage plane determination in early amphibian and fish embryos. Curr Biol 20(22):2040–2045. https://doi.org/10.1016/j.cub.2010.10.024

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Nieuwkoop PD, Faber J (1956) Normal table of Xenopus laevis (Daudin). A systematical and chronological survey of the development from the fertilized egg till the end of metamorphosis, vol 22. Garland Pub., New York, p 75

    Google Scholar 

  30. Mayor R, Morgan R, Sargent MG (1995) Induction of the prospective neural crest of Xenopus. Development 121(3):767–777

    CAS  PubMed  Google Scholar 

  31. Vize P (2011) Inducing egg laying via hCG injection (Vize lab). http://wiki.xenbase.org/xenwiki/index.php/Inducing_egg_laying_via_hCG_injection_(Vize_lab)

  32. VGP (2005) In vitro fertilization of Xenopus laevis (Conlon lab). http://wiki.xenbase.org/xenwiki/index.php/In_vitro_fertilization_of_Xenopus_laevis_(Conlon_lab)

  33. VGP (2011) Dejellying embryos (Zorn lab). http://wiki.xenbase.org/xenwiki/index.php/Dejellying_embryos_(Zorn_lab)

  34. Sive HL, Grainger RM, Harland RM (2007) Dejellying Xenopus laevis embryos. CSH Protoc 2007:pdb prot4731. https://doi.org/10.1101/pdb.prot4731

    Article  PubMed  Google Scholar 

  35. Paddock SW, Eliceiri KW (2014) Laser scanning confocal microscopy: history, applications, and related optical sectioning techniques. Methods Mol Biol 1075:9–47. https://doi.org/10.1007/978-1-60761-847-8_2

    Article  PubMed  Google Scholar 

  36. Ulrich M (2015) Confocal laser scanning microscopy. Hautarzt 66(7):504–510. https://doi.org/10.1007/s00105-015-3632-y

    Article  CAS  PubMed  Google Scholar 

  37. Gard DL (1999) Confocal microscopy and 3-D reconstruction of the cytoskeleton of Xenopus oocytes. Microsc Res Tech 44(6):388–414. https://doi.org/10.1002/(SICI)1097-0029(19990315)44:6<388::AID-JEMT2>3.0.CO;2-L

    Article  CAS  PubMed  Google Scholar 

  38. Nguyen PA, Groen AC, Loose M, Ishihara K, Wühr M, Field CM, Mitchison TJ (2014) Spatial organization of cytokinesis signaling reconstituted in a cell-free system. Science 346(6206):244–247. https://doi.org/10.1126/science.1256773

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Field CM, Groen AC, Nguyen PA, Mitchison TJ (2015) Spindle-to-cortex communication in cleaving, polyspermic Xenopus eggs. Mol Biol Cell 26(20):3628–3640. https://doi.org/10.1091/mbc.E15-04-0233

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Wühr M, Freeman RM Jr, Presler M, Horb ME, Peshkin L, Gygi SP, Kirschner MW (2014) Deep proteomics of the Xenopus laevis egg using an mRNA-derived reference database. Curr Biol 24(13):1467–1475. https://doi.org/10.1016/j.cub.2014.05.044

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Groen AC, Mitchison TJ (2016) Purification and fluorescent labeling of tubulin from Xenopus laevis egg extracts. Methods Mol Biol 1413:35–45. https://doi.org/10.1007/978-1-4939-3542-0_3

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

We thank Sean Megason, Angela DePace, Evangelos Gatzogiannis, Mike Levine, and Laurence Lemaire for usage of their microscopes. Thanks to members of the Wühr Lab for comments on the manuscript. This work was supported by grants GM39565 and 1R35GM128813 from the National Institutes of Health and Princeton University startup funding.

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Authors and Affiliations

  1. Lewis-Sigler Institute for Integrative Genomics and Department of Molecular Biology, Princeton University, Princeton, NJ, USA

    Thao Nguyen & Martin Wühr

  2. Department of Systems Biology, Harvard Medical School, Boston, MA, USA

    Timothy J. Mitchison

Authors
  1. Thao Nguyen
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  2. Timothy J. Mitchison
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  3. Martin Wühr
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Corresponding author

Correspondence to Martin Wühr .

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Editors and Affiliations

  1. Laboratory of Genetics, University of Wisconsin–Madison, Madison, WI, USA

    Francisco J. Pelegri

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Nguyen, T., Mitchison, T.J., Wühr, M. (2019). Immunofluorescence of Microtubule Assemblies in Amphibian Oocytes and Early Embryos. In: Pelegri, F. (eds) Vertebrate Embryogenesis. Methods in Molecular Biology, vol 1920. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9009-2_2

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  • DOI: https://doi.org/10.1007/978-1-4939-9009-2_2

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9008-5

  • Online ISBN: 978-1-4939-9009-2

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