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Whole-Mount BrdU Staining with Fluorescence In Situ Hybridization in Planarians

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Planarian Regeneration

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1774))

Abstract

Detection of cell proliferation based on the incorporation of 5′-bromo-2′-deoxyuridine (BrdU) has become a standard approach for studying stem cell and progenitor cell populations in developing and adult tissue. In this chapter, we describe three BrdU administration methods for planarians and a staining protocol combining BrdU detection with whole-mount fluorescence in situ hybridization (FISH). Collectively, these protocols enable the combined analysis of BrdU-incorporation and endogenous gene expression, as for example during lineage tracing applications.

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References

  1. Bravo R, Frank R, Blundell PA, Macdonald-Bravo H (1987) Cyclin/PCNA is the auxiliary protein of DNA polymerase-delta. Nature 326(6112):515–517. https://doi.org/10.1038/326515a0

    Article  CAS  PubMed  Google Scholar 

  2. Schluter C, Duchrow M, Wohlenberg C, Becker MH, Key G, Flad HD, Gerdes J (1993) The cell proliferation-associated antigen of antibody Ki-67: a very large, ubiquitous nuclear protein with numerous repeated elements, representing a new kind of cell cycle-maintaining proteins. J Cell Biol 123(3):513–522

    Article  CAS  PubMed  Google Scholar 

  3. Hendzel MJ, Wei Y, Mancini MA, Van Hooser A, Ranalli T, Brinkley BR, Bazett-Jones DP, Allis CD (1997) Mitosis-specific phosphorylation of histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation. Chromosoma 106(6):348–360

    Article  CAS  PubMed  Google Scholar 

  4. Taylor JH (1953) Intracellular localization of labeled nucleic acid determined by autoradiographs. Science 118(3071):555–557

    Article  CAS  PubMed  Google Scholar 

  5. Messier B, Leblond CP, Smart I (1958) Presence of DNA synthesis and mitosis in the brain of young adult mice. Exp Cell Res 14(1):224–226

    Article  CAS  PubMed  Google Scholar 

  6. Altman J (1969) Autoradiographic and histological studies of postnatal neurogenesis. IV. Cell proliferation and migration in the anterior forebrain, with special reference to persisting neurogenesis in the olfactory bulb. J Comp Neurol 137(4):433–457. https://doi.org/10.1002/cne.901370404

    Article  CAS  PubMed  Google Scholar 

  7. Potten CS, Hume WJ, Reid P, Cairns J (1978) The segregation of DNA in epithelial stem cells. Cell 15(3):899–906

    Article  CAS  PubMed  Google Scholar 

  8. Cotsarelis G, Sun TT, Lavker RM (1990) Label-retaining cells reside in the bulge area of pilosebaceous unit: implications for follicular stem cells, hair cycle, and skin carcinogenesis. Cell 61(7):1329–1337

    Article  CAS  PubMed  Google Scholar 

  9. Gratzner H (1982) Monoclonal antibody to 5-bromo- and 5-iododeoxyuridine: a new reagent for detection of DNA replication. Science (New York, NY) 218(4571):474–475

    Article  CAS  Google Scholar 

  10. Gonchoroff NJ, Katzmann JA, Currie RM, Evans EL, Houck DW, Kline BC, Greipp PR, Loken MR (1986) S-phase detection with an antibody to bromodeoxyuridine. Role of DNase pretreatment. J Immunol Methods 93(1):97–101

    Article  CAS  PubMed  Google Scholar 

  11. Manders EM, Stap J, Brakenhoff GJ, van Driel R, Aten JA (1992) Dynamics of three-dimensional replication patterns during the S-phase, analysed by double labelling of DNA and confocal microscopy. J Cell Sci 103(Pt 3):857–862

    CAS  PubMed  Google Scholar 

  12. Aten JA, Bakker PJ, Stap J, Boschman GA, Veenhof CH (1992) DNA double labelling with IdUrd and CldUrd for spatial and temporal analysis of cell proliferation and DNA replication. Histochem J 24(5):251–259

    Article  CAS  PubMed  Google Scholar 

  13. Salic A, Mitchison TJ (2008) A chemical method for fast and sensitive detection of DNA synthesis in vivo. Proc Natl Acad Sci U S A 105(7):2415–2420. https://doi.org/10.1073/pnas.0712168105

    Article  PubMed  PubMed Central  Google Scholar 

  14. Newmark PA, Sanchez Alvarado A (2000) Bromodeoxyuridine specifically labels the regenerative stem cells of planarians. Dev Biol 220(2):142–153. https://doi.org/10.1006/dbio.2000.9645

    Article  CAS  PubMed  Google Scholar 

  15. Reddien PW, Oviedo NJ, Jenkin JC, Sánchez Alvarado A (2005) SMEDWI-2 is a PIWI-like protein that regulates planarian stem cells. Science (New York, NY) 310(5752):1327–1330. https://doi.org/10.1126/science.1116110

    Article  CAS  Google Scholar 

  16. Eisenhoffer G, Kang H, Sanchez Alvarado A (2008) Molecular analysis of stem cells and their descendants during cell turnover and regeneration in the planarian Schmidtea mediterranea. Cell Stem Cell 3(3):327–339. https://doi.org/10.1016/j.stem.2008.07.002

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Wagner DE, Wang IE, Reddien PW (2011) Clonogenic neoblasts are pluripotent adult stem cells that underlie planarian regeneration. Science 332(6031):811–816. https://doi.org/10.1126/science.1203983

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Forsthoefel DJ, Park AE, Newmark PA (2011) Stem cell-based growth, regeneration, and remodeling of the planarian intestine. Dev Biol 356(2):445–459. https://doi.org/10.1016/j.ydbio.2011.05.669

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Cowles MW, Hubert A, Zayas RM (2012) A Lissencephaly-1 homologue is essential for mitotic progression in the planarian Schmidtea mediterranea. Dev Dyn 241(5):901–910. https://doi.org/10.1002/dvdy.23775

    Article  CAS  PubMed  Google Scholar 

  20. Pearson B, Eisenhoffer G, Gurley K, Rink J, Miller D, Sanchez Alvarado A (2009) Formaldehyde-based whole-mount in situ hybridization method for planarians. Dev Dyn 238(2):443–450. https://doi.org/10.1002/dvdy.21849

    Article  PubMed  PubMed Central  Google Scholar 

  21. King RS, Newmark PA (2013) In situ hybridization protocol for enhanced detection of gene expression in the planarian Schmidtea mediterranea. BMC Dev Biol 13:8. https://doi.org/10.1186/1471-213X-13-8

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Schurmann W, Peter R (2001) Planarian cell culture: a comparitive review of methods and an improved protocol for primary cultures of neoblasts. Belg J Zool 131(1):123–130

    Google Scholar 

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Acknowledgment

We would like to thank Adler C. and Vu H. for their help with optimization of the protocol. This work is supported by HHMI and Stowers Institute and NIH R37GM057260 to ASA.

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Authors and Affiliations

  1. The Scripps Research Institute, La Jolla, CA, USA

    Li-Chun Cheng

  2. Howard Hughes Medical Institute, Stowers Institute for Medical Research, Kansas City, MO, USA

    Alejandro Sánchez Alvarado

Authors
  1. Li-Chun Cheng
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  2. Alejandro Sánchez Alvarado
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Corresponding author

Correspondence to Alejandro Sánchez Alvarado .

Editor information

Editors and Affiliations

  1. Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany

    Jochen C. Rink

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Cheng, LC., Alvarado, A.S. (2018). Whole-Mount BrdU Staining with Fluorescence In Situ Hybridization in Planarians. In: Rink, J. (eds) Planarian Regeneration. Methods in Molecular Biology, vol 1774. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7802-1_15

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  • DOI: https://doi.org/10.1007/978-1-4939-7802-1_15

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7800-7

  • Online ISBN: 978-1-4939-7802-1

  • eBook Packages: Springer Protocols

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