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Analysis of Gene Expression Patterns and RNA Localization by Fluorescence in Situ Hybridization in Whole Mount Drosophila Testes

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RNA-Protein Complexes and Interactions

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2666))

Abstract

Researchers have used RNA in situ hybridization to detect the presence of RNA in cells and tissues for approximately 50 years. The recent development of a method capable of visualizing a single RNA molecule by utilizing tiled fluorescently labeled oligonucleotide probes that together produce a diffraction-limited spot has greatly increased the resolution of this technique, allowing for the precise determination of subcellular RNA localization and relative abundance. Here, we present our method for single molecule RNA fluorescence in situ hybridization (smFISH) in whole mount Drosophila testes and discuss how we have utilized this method to better understand the expression pattern of the highly unusual Y-linked genes.

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References

  1. Gall JG, Pardue ML (1969) Formation and detection of RNA-DNA hybrid molecules in cytological preparations. Proc Natl Acad Sci U S A 63(2):378–383. https://doi.org/10.1073/pnas.63.2.378

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Harrison PR, Conkie D, Paul J, Jones K (1973) Localisation of cellular globin messenger RNA by in situ hybridisation to complementary DNA. FEBS Lett 32(1):109–112. https://doi.org/10.1016/0014-5793(73)80749-5

    Article  CAS  PubMed  Google Scholar 

  3. Singer RH, Ward DC (1982) Actin gene expression visualized in chicken muscle tissue culture by using in situ hybridization with a biotinated nucleotide analog. Proc Natl Acad Sci U S A 79(23):7331–7335. https://doi.org/10.1073/pnas.79.23.7331

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Femino AM, Fay FS, Fogarty K, Singer RH (1998) Visualization of single RNA transcripts in situ. Science 280(5363):585–590. https://doi.org/10.1126/science.280.5363.585

    Article  CAS  PubMed  Google Scholar 

  5. Raj A, Tyagi S (2010) Detection of individual endogenous RNA transcripts in situ using multiple singly labeled probes. Methods Enzymol 472:365–386. https://doi.org/10.1016/S0076-6879(10)72004-8

    Article  CAS  PubMed  Google Scholar 

  6. Raj A, van den Bogaard P, Rifkin SA, van Oudenaarden A, Tyagi S (2008) Imaging individual mRNA molecules using multiple singly labeled probes. Nat Methods 5(10):877–879. https://doi.org/10.1038/nmeth.1253

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Kerstens HM, Poddighe PJ, Hanselaar AG (1995) A novel in situ hybridization signal amplification method based on the deposition of biotinylated tyramine. J Histochem Cytochem 43(4):347–352. https://doi.org/10.1177/43.4.7897179

    Article  CAS  PubMed  Google Scholar 

  8. Larsson C, Grundberg I, Soderberg O, Nilsson M (2010) In situ detection and genotyping of individual mRNA molecules. Nat Methods 7(5):395–397. https://doi.org/10.1038/nmeth.1448

    Article  CAS  PubMed  Google Scholar 

  9. Pare A, Lemons D, Kosman D, Beaver W, Freund Y, McGinnis W (2009) Visualization of individual Scr mRNAs during Drosophila embryogenesis yields evidence for transcriptional bursting. Curr Biol 19(23):2037–2042. https://doi.org/10.1016/j.cub.2009.10.028

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Lu J, Tsourkas A (2009) Imaging individual microRNAs in single mammalian cells in situ. Nucleic Acids Res 37(14):e100. https://doi.org/10.1093/nar/gkp482

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Fingerhut JM, Moran JV, Yamashita YM (2019) Satellite DNA-containing gigantic introns in a unique gene expression program during Drosophila spermatogenesis. PLoS Genet 15(5):e1008028. https://doi.org/10.1371/journal.pgen.1008028

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Fingerhut JM, Yamashita YM (2020) mRNA localization mediates maturation of cytoplasmic cilia in Drosophila spermatogenesis. J Cell Biol 219(9). https://doi.org/10.1083/jcb.202003084

  13. Medioni C, Mowry K, Besse F (2012) Principles and roles of mRNA localization in animal development. Development 139(18):3263–3276. https://doi.org/10.1242/dev.078626

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Kumano G (2012) Polarizing animal cells via mRNA localization in oogenesis and early development. Develop Growth Differ 54(1):1–18. https://doi.org/10.1111/j.1440-169X.2011.01301.x

    Article  CAS  Google Scholar 

  15. Broadus J, Fuerstenberg S, Doe CQ (1998) Staufen-dependent localization of prospero mRNA contributes to neuroblast daughter-cell fate. Nature 391(6669):792–795. https://doi.org/10.1038/35861

    Article  CAS  PubMed  Google Scholar 

  16. Li P, Yang X, Wasser M, Cai Y, Chia W (1997) Inscuteable and Staufen mediate asymmetric localization and segregation of prospero RNA during Drosophila neuroblast cell divisions. Cell 90(3):437–447. https://doi.org/10.1016/s0092-8674(00)80504-8

    Article  CAS  PubMed  Google Scholar 

  17. Lawrence JB, Singer RH (1986) Intracellular localization of messenger RNAs for cytoskeletal proteins. Cell 45(3):407–415. https://doi.org/10.1016/0092-8674(86)90326-0

    Article  CAS  PubMed  Google Scholar 

  18. Mingle LA, Okuhama NN, Shi J, Singer RH, Condeelis J, Liu G (2005) Localization of all seven messenger RNAs for the actin-polymerization nucleator Arp2/3 complex in the protrusions of fibroblasts. J Cell Sci 118(Pt 11):2425–2433. https://doi.org/10.1242/jcs.02371

    Article  CAS  PubMed  Google Scholar 

  19. Lee C, Occhipinti P, Gladfelter AS (2015) PolyQ-dependent RNA-protein assemblies control symmetry breaking. J Cell Biol 208(5):533–544. https://doi.org/10.1083/jcb.201407105

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Trcek T, Grosch M, York A, Shroff H, Lionnet T, Lehmann R (2015) Drosophila germ granules are structured and contain homotypic mRNA clusters. Nat Commun 6:7962. https://doi.org/10.1038/ncomms8962

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Niepielko MG, Eagle WVI, Gavis ER (2018) Stochastic seeding coupled with mRNA self-recruitment generates heterogeneous Drosophila germ granules. Curr Biol 28(12):1872–1881. e1873. https://doi.org/10.1016/j.cub.2018.04.037

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Moon SL, Morisaki T, Khong A, Lyon K, Parker R, Stasevich TJ (2019) Multicolour single-molecule tracking of mRNA interactions with RNP granules. Nat Cell Biol 21(2):162–168. https://doi.org/10.1038/s41556-018-0263-4

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Khong A, Matheny T, Jain S, Mitchell SF, Wheeler JR, Parker R (2017) The stress granule transcriptome reveals principles of mRNA accumulation in stress granules. Mol Cell 68(4):808–820 e805. https://doi.org/10.1016/j.molcel.2017.10.015

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Kwon S (2013) Single-molecule fluorescence in situ hybridization: quantitative imaging of single RNA molecules. BMB Rep 46(2):65–72. https://doi.org/10.5483/bmbrep.2013.46.2.016

    Article  PubMed  PubMed Central  Google Scholar 

  25. Fuller MT (1993) Spermatogenesis. In: Bate M, Arias AM (eds) The development of Drosophila melanogaster, vol 1. Cold Spring Harbor Laboratory Press, New York, pp 71–148

    Google Scholar 

  26. Hardy RW, Tokuyasu KT, Lindsley DL (1981) Analysis of spermatogenesis in Drosophila melanogaster bearing deletions for Y-chromosome fertility genes. Chromosoma 83(5):593–617

    Article  CAS  PubMed  Google Scholar 

  27. Goldstein LS, Hardy RW, Lindsley DL (1982) Structural genes on the Y chromosome of Drosophila melanogaster. Proc Natl Acad Sci U S A 79(23):7405–7409

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Carvalho AB, Lazzaro BP, Clark AG (2000) Y chromosomal fertility factors kl-2 and kl-3 of Drosophila melanogaster encode dynein heavy chain polypeptides. Proc Natl Acad Sci U S A 97(24):13239–13244. https://doi.org/10.1073/pnas.230438397

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Bonaccorsi S, Pisano C, Puoti F, Gatti M (1988) Y chromosome loops in Drosophila melanogaster. Genetics 120(4):1015–1034

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Gatti MP, S. (1983) Cytological and genetic analysis of the Y chromosome of Drosophila melanogaster. Chromosoma 88:349–373. https://doi.org/10.1007/BF00285858

    Article  Google Scholar 

  31. Pimpinelli S, Bonaceeorsi J, Gatti M, Sandler L (1986) The peculiar genetic organization of Drosophila heterochromatin. Trends Genet 2:17–20

    Article  Google Scholar 

  32. Lohe AR, Hilliker AJ, Roberts PA (1993) Mapping simple repeated DNA sequences in heterochromatin of Drosophila melanogaster. Genetics 134(4):1149–1174

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Hoskins RA, Smith CD, Carlson JW, Carvalho AB, Halpern A, Kaminker JS, Kennedy C, Mungall CJ, Sullivan BA, Sutton GG, Yasuhara JC, Wakimoto BT, Myers EW, Celniker SE, Rubin GM, Karpen GH (2002) Heterochromatic sequences in a Drosophila whole-genome shotgun assembly. Genome Biol 3(12):RESEARCH0085

    Article  PubMed  PubMed Central  Google Scholar 

  34. Carvalho AB (2002) Origin and evolution of the Drosophila Y chromosome. Curr Opin Genet Dev 12(6):664–668

    Article  CAS  PubMed  Google Scholar 

  35. Peacock WJ, Lohe AR, Gerlach WL, Dunsmuir P, Dennis ES, Appels R (1978) Fine structure and evolution of DNA in heterochromatin. Cold Spring Harb Symp Quant Biol 42(Pt 2):1121–1135

    Article  CAS  PubMed  Google Scholar 

  36. Chandley AC, Bateman AJ (1962) Timing of spermatogenesis in Drosophila melanogaster using tritiated thymidine. Nature 193:299–300

    Article  CAS  PubMed  Google Scholar 

  37. Tates AD (1971) Cytodifferentiation during spermatogenesis in Drosophila melanogaster: an electon microsope study. Rijksuniversiteit, Leiden

    Google Scholar 

  38. Tokuyasu KT (1975) Dynamics of spermiogenesis in Drosophila melanogaster. VI. Significance of "onion" nebenkern formation. J Ultrastruct Res 53(1):93–112. https://doi.org/10.1016/s0022-5320(75)80089-x

    Article  CAS  PubMed  Google Scholar 

  39. Warsinger-Pepe N, Li D, Yamashita YM (2020) Regulation of nucleolar dominance in Drosophila melanogaster. Genetics 214(4):991–1004. https://doi.org/10.1534/genetics.119.302471

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Lu KL, Nelson JO, Watase GJ, Warsinger-Pepe N, Yamashita YM (2018) Transgenerational dynamics of rDNA copy number in Drosophila male germline stem cells. elife 7. https://doi.org/10.7554/eLife.32421

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Author information

Authors and Affiliations

  1. Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Department of Biology, Cambridge, MA, USA

    Jaclyn M. Fingerhut & Yukiko M. Yamashita

  2. Howard Hughes Medical Institute, Cambridge, MA, USA

    Jaclyn M. Fingerhut & Yukiko M. Yamashita

Authors
  1. Jaclyn M. Fingerhut
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  2. Yukiko M. Yamashita
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Corresponding authors

Correspondence to Jaclyn M. Fingerhut or Yukiko M. Yamashita .

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Editors and Affiliations

  1. Beckman Research Institute, Center for RNA Biology and Therapeutics, Irell & Manella Graduate School of Biological Sciences, Duarte, CA, USA

    Ren-Jang Lin

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Fingerhut, J.M., Yamashita, Y.M. (2023). Analysis of Gene Expression Patterns and RNA Localization by Fluorescence in Situ Hybridization in Whole Mount Drosophila Testes. In: Lin, RJ. (eds) RNA-Protein Complexes and Interactions. Methods in Molecular Biology, vol 2666. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3191-1_2

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  • DOI: https://doi.org/10.1007/978-1-0716-3191-1_2

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3190-4

  • Online ISBN: 978-1-0716-3191-1

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