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Flow Cytometric Quantification, Isolation, and Subsequent Epigenetic Analysis of Tetraploid Neurons

  • Noelia López-Sánchez
  • Iris Patiño-Parrado
  • José María FradeEmail author
Protocol
Part of the Neuromethods book series (NM, volume 131)

Abstract

Different forms of genomic mosaicism can be detected in vertebrate neurons, including copy number variation, L1 transposition, and aneuploidy. In addition, some populations of vertebrate neurons can also show double the normal amount of DNA, a condition referred to as somatic tetraploidy. These neurons are generated during early stages of development, as they migrate to their adult locations in the adult nervous system, and constitute subpopulations of projection neurons. We have previously shown that neuronal tetraploidy can be characterized by flow cytometry using isolated cell nuclei from different mammalian and avian structures. In this chapter, we describe this procedure using a different model system: the rhombencephalic derivatives from adult zebrafish. In addition, tetraploid neuronal nuclei can be isolated by fluorescence-activated cell sorting and their genomic DNA used for further analyses, either used directly or after whole-genome amplification. Here we show as an example how to perform epigenetic analyses to characterize CpG methylation in differentially methylated regions controlling the Rasgrf1-imprinting domain in mice.

Key words

Cell sorting FACS Tetraploid neuron Cell nuclei Zebrafish Genomic imprinting DNA methylation Pyrosequencing 

Notes

Acknowledgments

We thank Gonzalo García de Polavieja and Aixa Morales for the zebrafish specimens used in this study. This research was supported by the Ministerio de Economía y Competitividad (grant numbers SAF2015-68488-R) and a R&D contract with Tetraneuron S.L.

References

  1. 1.
    Masterson J (1994) Stomatal size in fossil plants: evidence for polyploidy in majority of angiosperms. Science 264:421–424CrossRefPubMedGoogle Scholar
  2. 2.
    Sugimoto-Shirasu K, Roberts K (2003) "big it up": endoreduplication and cell-size control in plants. Curr Opin Plant Biol 6:544–553CrossRefPubMedGoogle Scholar
  3. 3.
    Madlung A, Wendel JF (2013) Genetic and epigenetic aspects of polyploid evolution in plants. Cytogenet Genome Res 140:270–285CrossRefPubMedGoogle Scholar
  4. 4.
    Otto SP, Whitton J (2000) Polyploid incidence and evolution. Annu Rev Genet 34:401–437CrossRefPubMedGoogle Scholar
  5. 5.
    Li S, Yin L, Cole ES, Udani RA, Karrer KM (2006) Progeny of germ line knockouts of ASI2, a gene encoding a putative signal transduction receptor in Tetrahymena Thermophila, fail to make the transition from sexual reproduction to vegetative growth. Dev Biol 295:633–646CrossRefPubMedGoogle Scholar
  6. 6.
    Kolics B, Ács Z, Chobanov DP, Orci KM, Qiang LS, Kovács B, Kondorosy E, Decsi K, Taller J, Specziár A, Orbán L, Müller T (2012) Re-visiting phylogenetic and taxonomic relationships in the genus Saga (Insecta: Orthoptera). PLoS One 7:e42229CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Leggatt RA, Iwama GK (2003) Occurrence of polyploidy in the fishes. Rev Fish Biol Fish 13:237–246CrossRefGoogle Scholar
  8. 8.
    Vernon JA, Butsch J (1957) Effect of tetraploidy on learning and retention in the salamander. Science 125:1033–1034CrossRefPubMedGoogle Scholar
  9. 9.
    Szaro BG, Tompkins R (1987) Effect of tetraploidy on dendritic branching in neurons and glial cells of the frog, Xenopus laevis. J Comp Neurol 258:304–316CrossRefPubMedGoogle Scholar
  10. 10.
    Trifonov VA, Paoletti A, Caputo Barucchi V, Kalinina T, O’Brien PC, Ferguson-Smith MA, Giovannotti M (2015) Comparative chromosome painting and NOR distribution suggest a complex hybrid origin of triploid Lepidodactylus lugubris (Gekkonidae). PLoS One 10:e0132380CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Frawley LE, Orr-Weaver TL (2015) Polyploidy. Curr Biol 25:R353–R358CrossRefPubMedGoogle Scholar
  12. 12.
    Biesterfeld S, Gerres K, Fischer-Wein G, Böcking A (1994) Polyploidy in non-neoplastic tissues. J Clin Pathol 47:38–42CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Edgar BA, Orr-Weaver TL (2001) Endoreplication cell cycles: more for less. Cell 105:297–306CrossRefPubMedGoogle Scholar
  14. 14.
    Ullah Z, Lee CY, Depamphilis ML (2009) Cip/kip cyclin-dependent protein kinase inhibitors and the road to polyploidy. Cell Div 4:10Google Scholar
  15. 15.
    Ullah Z, Lee CY, Lilly MA, DePamphilis ML (2009) Developmentally programmed endoreduplication in animals. Cell Cycle 8:1501–1509CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Pandit SK, Westendorp B, de Bruin A (2013) Physiological significance of polyploidization in mammalian cells. Trends Cell Biol 23:556–566CrossRefPubMedGoogle Scholar
  17. 17.
    Unhavaithaya Y, Orr-Weaver TL (2012) Polyploidization of glia in neural development links tissue growth to blood-brain barrier integrity. Genes Dev 26:31–36CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Lilly MA, Spradling AC (1996) The drosophila endocycle is controlled by cyclin E and lacks a checkpoint ensuring S-phase completion. Genes Dev 10:2514–2526CrossRefPubMedGoogle Scholar
  19. 19.
    Buntrock L, Marec F, Krueger S, Traut W (2012) Organ growth without cell division: somatic polyploidy in a moth, Ephestia kuehniella. Genome 55:755–763CrossRefPubMedGoogle Scholar
  20. 20.
    Vitrat N, Cohen-Solal K, Pique C, Le Couedic JP, Norol F, Larsen AK, Katz A, Vainchenker W, Debili N (1998) Endomitosis of human megakaryocytes are due to abortive mitosis. Blood 91:3711–3723PubMedGoogle Scholar
  21. 21.
    Ullah Z, Kohn MJ, Yagi R, Vassilev LT, DePamphilis ML (2008) Differentiation of trophoblast stem cells into giant cells is triggered by p57/Kip2 inhibition of CDK1 activity. Genes Dev 22:3024–3036CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Vinogradov AE, Anatskaya OV, Kudryavtsev BN (2001) Relationship of hepatocyte ploidy levels with body size and growth rate in mammals. Genome 44:350–360CrossRefPubMedGoogle Scholar
  23. 23.
    Zanet J, Freije A, Ruiz M, Coulon V, Sanz JR, Chiesa J, Gandarillas A (2010) A mitosis block links active cell cycle with human epidermal differentiation and results in endoreplication. PLoS One 5:e15701CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Nagata Y, Jones MR, Nguyen HG, McCrann DJ, St Hilaire C, Schreiber BM, Hashimoto A, Inagaki M, Earnshaw WC, Todokoro K, Ravid K (2005) Vascular smooth muscle cell polyploidization involves changes in chromosome passenger proteins and an endomitotic cell cycle. Exp Cell Res 305:277–291CrossRefPubMedGoogle Scholar
  25. 25.
    Kellerman S, Moore JA, Zierhut W, Zimmer HG, Campbell J, Gerdes AM (1992) Nuclear DNA content and nucleation patterns in rat cardiac myocytes from different models of cardiac hypertrophy. J Mol Cell Cardiol 24:497–505CrossRefPubMedGoogle Scholar
  26. 26.
    Coggeshall RE, Yaksta BA, Swartz FJ (1970) A cytophotometric analysis of the DNA in the nucleus of the giant cell, R-2, in Aplysia. Chromosoma 32:205–212PubMedGoogle Scholar
  27. 27.
    Manfredi Romanini MG, Fraschini A, Bernocchi G (1973) DNA content and nuclear area in the neurons of the cerebral ganglion in Helix pomatia. Ann Histochim 18:49–58PubMedGoogle Scholar
  28. 28.
    Castelfranco AM, Hartline DK (2016) Evolution of rapid nerve conduction. Brain Res 1641(Pt a):11–33CrossRefPubMedGoogle Scholar
  29. 29.
    Lasek RJ, Dower WJ (1971) Aplysia californica: analysis of nuclear DNA in individual nuclei of giant neurons. Science 172:278–280CrossRefPubMedGoogle Scholar
  30. 30.
    Swift H (1953) Quantitative aspects of nuclear nucleoproteins. Int Rev Cytol 2:1–76CrossRefGoogle Scholar
  31. 31.
    Lapham LW (1968) Tetraploid DNA content of Purkinje neurons of human cerebellar cortex. Science 159:310–312CrossRefPubMedGoogle Scholar
  32. 32.
    Mosch B, Morawski M, Mittag A, Lenz D, Tarnok A, Arendt T (2007) Aneuploidy and DNA replication in the normal human brain and Alzheimer’s disease. J Neurosci 27:6859–6867CrossRefPubMedGoogle Scholar
  33. 33.
    Morillo SM, Escoll P, de la Hera A, Frade JM (2010) Somatic tetraploidy in specific chick retinal ganglion cells induced by nerve growth factor. Proc Natl Acad Sci U S A 107:109–114CrossRefPubMedGoogle Scholar
  34. 34.
    Shirazi Fard S, Jarrin M, Boije H, Fillon V, All-Eriksson C, Hallböök F (2013) Heterogenic final cell cycle by chicken retinal Lim1 horizontal progenitor cells leads to heteroploid cells with a remaining replicated genome. PLoS One 8:e59133CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Shirazi Fard S, All-Ericsson C, Hallböök F (2014) The heterogenic final cell cycle of chicken retinal Lim1 horizontal cells is not regulated by the DNA damage response pathway. Cell Cycle 13:408–417CrossRefPubMedGoogle Scholar
  36. 36.
    López-Sánchez N, Frade JM (2013) Genetic evidence for p75NTR-dependent tetraploidy in cortical projection neurons from adult mice. J Neurosci 33:7488–7500CrossRefPubMedGoogle Scholar
  37. 37.
    Ovejero-Benito MC, Frade JM (2013) Brain-derived neurotrophic factor-dependent cdk1 inhibition prevents G2/M progression in differentiating tetraploid neurons. PLoS One 8:e64890CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Ovejero-Benito MC, Frade JM (2015) p27Kip1 participates in the regulation of endoreplication in differentiating chick retinal ganglion cells. Cell Cycle 14:2311–2322CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Kingsbury MA, Yung YC, Peterson SE, Westra JW, Chun J (2006) Aneuploidy in the normal and diseased brain. Cell Mol Life Sci 63:2626–2641CrossRefPubMedGoogle Scholar
  40. 40.
    Ying QL, Nichols J, Evans EP, Smith AG (2002) Changing potency by spontaneous fusion. Nature 416:545–548CrossRefPubMedGoogle Scholar
  41. 41.
    Feulgen R, Rossenbeck H (1924) Mikroskopisch-chemischer Nachweis einer Nukleinsäure von Typus der Thymonukleinsäure und die darauf beruhende selektive Färbung von Zellkernen in mikroskopischen Präparaten. Hoppe Seylers Z Physiol Chem 135:203–248CrossRefGoogle Scholar
  42. 42.
    Chieco P, Derenzini M (1999) The Feulgen reaction 75 years on. Histochem Cell Biol 111:345–358CrossRefPubMedGoogle Scholar
  43. 43.
    Woodard J, Gorovsky M, Swift H (1966) DNA content of a chromosome of Trillium erectum: effect of cold treatment. Science 151:215–216CrossRefPubMedGoogle Scholar
  44. 44.
    Herman CJ, Lapham LW (1968) DNA content of neurons in the cat hippocampus. Science 160:537CrossRefPubMedGoogle Scholar
  45. 45.
    Herman CJ, Lapham LW (1969) Neuronal polyploidy and nuclear volumes in the cat central nervous system. Brain Res 15:35–48CrossRefPubMedGoogle Scholar
  46. 46.
    Museridze DP, Svanidze IK, Macharashvili DN (1975) Content of DNA and dry weight of the nuclei of neurons of the external geniculate body and retina of the eye in guinea pigs. Sov J Dev Biol 5:269–272PubMedGoogle Scholar
  47. 47.
    Swartz FJ, Bhatnagar KP (1981) Are CNS neurons polyploid? A critical analysis based upon cytophotometric study of the DNA content of cerebellar and olfactory bulbar neurons of the bat. Brain Res 208:267–281CrossRefPubMedGoogle Scholar
  48. 48.
    Mosch B, Mittag A, Lenz D, Arendt T, Tárnok A (2006) Laser scanning cytometry in human brain slices. Cytometry A 69:135–138CrossRefPubMedGoogle Scholar
  49. 49.
    Fulwyler MJ (1965) Electronic separation of biological cells by volume. Science 150:910–911CrossRefPubMedGoogle Scholar
  50. 50.
    Kamentsky LA, Melamed MR, Derman H (1965) Spectrophotometer: new instrument for ultrarapid cell analysis. Science 150:630–631CrossRefPubMedGoogle Scholar
  51. 51.
    Van Dilla MA, Trujillo TT, Mullaney PF, Coulter JR (1969) Cell microfluorometry: a method for rapid fluorescence measurement. Science 163:1213–1214CrossRefPubMedGoogle Scholar
  52. 52.
    Hulett HR, Bonner WA, Barrett J, Herzenberg LA (1969) Cell sorting: automated separation of mammalian cells as a function of intracellular fluorescence. Science 166:747–749CrossRefPubMedGoogle Scholar
  53. 53.
    Hoshino T, Nomura K, Wilson CB, Knebel KD, Gray JW (1978) The distribution of nuclear DNA from human brain-tumor cells. J Neurosurg 49:13–21CrossRefPubMedGoogle Scholar
  54. 54.
    Bernocchi G, Barni S (1985) On the heterogeneity of Purkinje neurons in vertebrates. Cytochemical and morphological studies of chromatin during eel (Anguilla Anguilla L.) life cycle. J Hirnforsch 26:227–235PubMedGoogle Scholar
  55. 55.
    Nunez R (2001) DNA measurement and cell cycle analysis by flow cytometry. Curr Issues Mol Biol 3:67–70PubMedGoogle Scholar
  56. 56.
    Martinez-Morales PL, Quiroga AC, Barbas JA, Morales AV (2010) SOX5 controls cell cycle progression in neural progenitors by interfering with the WNT-beta-catenin pathway. EMBO Rep 11:466–472CrossRefPubMedPubMedCentralGoogle Scholar
  57. 57.
    López-Sánchez N, Ovejero-Benito MC, Borreguero L, Frade JM (2011) Control of neuronal ploidy during vertebrate development. Results Probl Cell Differ 53:547–563CrossRefPubMedGoogle Scholar
  58. 58.
    Hasbold J, Hodgkin PD (2000) Flow cytometric cell division tracking using nuclei. Cytometry 40(3):230–237CrossRefPubMedGoogle Scholar
  59. 59.
    Westra JW, Barral S, Chun J (2009) A reevaluation of tetraploidy in the Alzheimer’s disease brain. Neurodegener Dis 6:221–229CrossRefPubMedPubMedCentralGoogle Scholar
  60. 60.
    Westra JW, Rivera RR, Bushman DM, Yung YC, Peterson SE, Barral S, Chun J (2010) Neuronal DNA content variation (DCV) with regional and individual differences in the human brain. J Comp Neurol 518:3981–4000CrossRefPubMedPubMedCentralGoogle Scholar
  61. 61.
    Nüsse M, Beisker W, Hoffmann C, Tarnok A (1990) Flow cytometric analysis of G1- and G2/M-phase subpopulations in mammalian cell nuclei using side scatter and DNA content measurements. Cytometry 11:813–821CrossRefPubMedGoogle Scholar
  62. 62.
    Darzynkiewicz Z, Traganos F, Kapuscinski J, Staiano-Coico L, Melamed MR (1984) Accessibility of DNA in situ to various fluorochromes: relationship to chromatin changes during erythroid differentiation of friend leukemia cells. Cytometry 5:355–363CrossRefPubMedGoogle Scholar
  63. 63.
    Darzynkiewicz Z (2011) Critical aspects in analysis of cellular DNA content. In: Robinson JP (ed) Current protocols in cytometry. Wiley, New YorkGoogle Scholar
  64. 64.
    López-Sánchez N, Frade JM (2013) Cell cycle analysis in the vertebrate brain using immunolabeled fresh cell nuclei. Bio-protocol 3:e973CrossRefGoogle Scholar
  65. 65.
    López-Sánchez N, Frade JM (2015) Flow cytometric analysis of DNA synthesis and apoptosis in central nervous system using fresh cell nuclei. Methods Mol Biol 1254:33–42CrossRefPubMedGoogle Scholar
  66. 66.
    Shapiro HM (2003) Practical flow cytometry, 4th edn. Wiley, New YorkCrossRefGoogle Scholar
  67. 67.
    Murciano A, Zamora J, López-Sánchez J, Frade JM (2002) Interkinetic nuclear movement may provide spatial clues to the regulation of neurogenesis. Mol Cell Neurosci 21:285–300CrossRefPubMedGoogle Scholar
  68. 68.
    Slaninová I, López-Sánchez N, Šebrlová K, Vymazal O, Frade JM, Táborská E (2016) Introduction of macarpine as a novel cell-permeant DNA dye for live cell imaging and flow cytometry sorting. Biol Cell 108:1–18CrossRefPubMedGoogle Scholar
  69. 69.
    Li JY, Lees-Murdock DJ, GL X, Walsh CP (2004) Timing of establishment of paternal methylation imprints in the mouse. Genomics 84:952–960CrossRefPubMedGoogle Scholar
  70. 70.
    Kumaki Y, Oda M, Okano M (2008) QUMA: quantification tool for methylation analysis. Nucleic Acids Res 36(suppl 2):W170–W175CrossRefPubMedPubMedCentralGoogle Scholar
  71. 71.
    Hutter KJ, Eipel HE (1978) Flow cytometric determinations of cellular substances in algae, bacteria, moulds and yeasts. Antonie Van Leeuwenhoek 44:269–282CrossRefPubMedGoogle Scholar
  72. 72.
    Gray JW, Carver JH, George YS, Mendelsohn ML (1977) Rapid cell cycle analysis by measurement of the radioactivity per cell in a narrow window in S phase (RCSi). Cell Tissue Kinet 10:97–109PubMedGoogle Scholar
  73. 73.
    Wilder ME, Cram LS (1977) Differential fluorochromasia of human lymphocytes as measured by flow cytometry. J Histochem Cytochem 25:888–891CrossRefPubMedGoogle Scholar
  74. 74.
    Van Dilla MA, Gledhill BL, Lake S, Dean PN, Gray JW, Kachel V, Barlogie B, Göhde W (1977) Measurement of mammalian sperm deoxyribonucleic acid by flow cytometry. Problems and approaches. J Histochem Cytochem 25:763–773CrossRefPubMedGoogle Scholar
  75. 75.
    Jensen RH (1977) Chromomycin A3 as a fluorescent probe for flow cytometry of human gynecologic samples. J Histochem Cytochem 25:573–579CrossRefPubMedGoogle Scholar
  76. 76.
    Pedersen T, Larsen JK, Krarup T (1978) Characterization of bladder tumours by flow cytometry on bladder washings. Eur Urol 4:351–355PubMedGoogle Scholar
  77. 77.
    Frederiksen P, Reske-Nielsen E, Bichel P (1978) Flow cytometry in tumours of the brain. Acta Neuropathol 41:179–183CrossRefPubMedGoogle Scholar
  78. 78.
    Morillo SM, Abanto EP, Román MJ, Frade JM (2012) Nerve growth factor-induced cell cycle reentry in newborn neurons is triggered by p38MAPK-dependent E2F4 phosphorylation. Mol Cell Biol 32:2722–2737Google Scholar
  79. 79.
    Arlotta P, Molyneaux BJ, Chen J, Inoue J, Kominami R, Macklis JD (2005) Neuronal subtype-specific genes that control corticospinal motor neuron development in vivo. Neuron 45:207–221CrossRefPubMedGoogle Scholar
  80. 80.
    Wan LB, Bartolomei MS (2008) Regulation of imprinting in clusters: noncoding RNAs versus insulators. Adv Genet 61:207–223PubMedGoogle Scholar
  81. 81.
    Leonhardt H, Page AW, Weier HU, Bestor TH (1992) A targeting sequence directs DNA methyltransferase to sites of DNA replication in mammalian nuclei. Cell 71:865–873CrossRefPubMedGoogle Scholar
  82. 82.
    Constância M, Pickard B, Kelsey G, Reik W (1998) Imprinting mechanisms. Genome Res 8:881–900CrossRefPubMedGoogle Scholar
  83. 83.
    Frommer M, McDonald LE, Millar DS, Collis CM, Watt F, Grigg GW, Molloy PL, Paul CL (1992) A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc Natl Acad Sci U S A 89:1827–1831CrossRefPubMedPubMedCentralGoogle Scholar
  84. 84.
    Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A 74:5463–5467CrossRefPubMedPubMedCentralGoogle Scholar
  85. 85.
    Ronaghi M, Karamohamed S, Pettersson B, Uhlén M, Nyrén P (1996) Real-time DNA sequencing using detection of pyrophosphate release. Anal Biochem 242:84–89CrossRefPubMedGoogle Scholar
  86. 86.
    Langaee T, Ronaghi M (2005) Genetic variation analyses by pyrosequencing. Mutat Res 573:96–102CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Noelia López-Sánchez
    • 1
    • 2
  • Iris Patiño-Parrado
    • 1
  • José María Frade
    • 1
    Email author
  1. 1.Department of Molecular, Cellular and Developmental NeurobiologyCajal Institute (IC-CSIC)MadridSpain
  2. 2.Tetraneuron S.L.ValenciaSpain

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