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In Situ Hybridization Methods pp 171–206Cite as

Manual and Automated Whole-Mount In Situ Hybridization for Systematic Gene Expression Analysis in Embryonic Zebrafish Forebrain

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Part of the book series: Neuromethods ((NM,volume 99))

Abstract

Large-scale data collections of gene expression patterns have been assembled to construct molecular atlases of embryonic and adult vertebrate brains. These open data collections are proving useful as profound resources for developmental, physiological, and functional studies and provide a basis for deciphering the molecular anatomies of vertebrate brains. The interrogation of gene expression data from such resources allows identifying genes with restricted regional patterns and neuronal profiles. The corresponding cDNA/EST clones can be ordered from publicly accessible clone collections strongly facilitating the systematic analysis of gene expression. However, in order to characterize the expression of a large number of genes, it is necessary to develop automated procedures that allow high-throughput analysis of samples. In this chapter, we describe our approach to analyze developmental regulatory genes and neuronal markers in the embryonic zebrafish brain by chromogenic whole-mount in situ hybridization (WISH) using manual and automated procedures. For automation, we make use of the InsituPro robot, which enables automated processing of up to 96 samples in parallel. We provide detailed step-by-step protocols of embryo collection, fixation and permeabilization, cDNA template and RNA probe preparation, manual and automated in situ hybridization, immunohistochemical detection and staining, and mounting and imaging of successfully stained specimen.

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References

  1. Yokoyama S, Ito Y, Ueno-Kudoh H, et al. (2009) A systems approach reveals that the myogenesis genome network is regulated by the transcriptional repressor RP58. Dev Cell 17(6):836–848

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  2. Magdaleno S, Jensen P, Brumwell CL, Seal A, Lehman K, Asbury A, Cheung T, Cornelius T, Batten DM, Eden C, Norland SM, Rice DS, Dosooye N, Shakya S, Mehta P, Curran T (2006) BGEM: an in situ hybridization database of gene expression in the embryonic and adult mouse nervous system. PLoS Biol 4(4):e86

    Article  PubMed Central  PubMed  Google Scholar 

  3. Visel A, Thaller C, Eichele G (2004) GenePaint.org: an atlas of gene expression patterns in the mouse embryo. Nucleic Acids Res 32(Database issue):D552–D556

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  4. Diez-Roux G, Banfi S, Sultan M, et al (2011) A high-resolution anatomical atlas of the transcriptome in the mouse embryo. PLoS Biol 9(1):e1000582

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  5. Eichele G, Diez-Roux G (2011) High-throughput analysis of gene expression on tissue sections by in situ hybridization. Methods 53(4):417–423

    Article  CAS  PubMed  Google Scholar 

  6. Bernard A, Lubbers LS, Tanis KQ et al (2012) Transcriptional architecture of the primate neocortex. Neuron 73(6):1083–1099

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  7. Lein ES, Hawrylycz MJ, Ao N et al (2007) Genome-wide atlas of gene expression in the adult mouse brain. Nature 445(7124):168–176

    Article  CAS  PubMed  Google Scholar 

  8. Zeng H, Shen EH, Hohmann JG et al (2012) Large-scale cellular-resolution gene profiling in human neocortex reveals species-specific molecular signatures. Cell 149(2):483–496

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  9. Puelles L, Harrison M, Paxinos G, Watson C (2013) A developmental ontology for the mammalian brain based on the prosomeric model. Trends Neurosci 36(10):570–578

    Article  CAS  PubMed  Google Scholar 

  10. Puelles L, Martínez-de-la-Torre M, Bardet SM, Rubenstein JLR (2012) Hypothalamus. In: Watson C, Paxinos G, Puelles L (eds) The mouse nervous system, 1st edn. Academic Press, Elsevier, Amsterdam, pp 221–312

    Chapter  Google Scholar 

  11. Puelles L, Martínez-de-la-Torre M, Bardet SM, Rubenstein JLR (2012) Diencephalon. In: Watson C, Paxinos G, Puelles L (eds) The mouse nervous system, 1st edn. Academic Press, Elsevier, Amsterdam, pp 313–336

    Chapter  Google Scholar 

  12. Shimogori T, Lee DA, Miranda-Angulo A, Yang Y, Wang H, Jiang L, Yoshida AC, Kataoka A, Mashiko H, Avetisyan M, Qi L, Qian J, Blackshaw S (2010) A genomic atlas of mouse hypothalamic development. Nat Neurosci 13(6):767–775

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  13. Medina L, Abellán A (2012) Subpallial structures. In: Watson C, Paxinos G, Puelles L (eds) The mouse nervous system, 1st edn. Academic Press, Elsevier, Amsterdam, pp 173–220

    Chapter  Google Scholar 

  14. Striedter GF, Belgard TG, Chen CC et al (2014) NSF workshop report: discovering general principles of nervous system organization by comparing brain maps across species. J Comp Neurol 522(7):1445–1453

    Article  PubMed  Google Scholar 

  15. Howe DG, Bradford YM, Conlin T et al (2013) ZFIN, the zebrafish model organism database: increased support for mutants and transgenics. Nucleic Acids Res 41(Database issue):D854–D860

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  16. Thisse B, Heyer V, Lux A, Alunni V, Degrave A, Seiliez I, Kirchner J, Parkhill JP, Thisse C (2004) Spatial and temporal expression of the zebrafish genome by large-scale in situ hybridization screening. Methods Cell Biol 77:505–519

    Article  CAS  PubMed  Google Scholar 

  17. Hauptmann G, Gerster T (1994) Two-color whole-mount in situ hybridization to vertebrate and Drosophila embryos. Trends Genet 10(8):266. doi:10.1016/0168-9525(90)90008-T

  18. Hauptmann G, Gerster T (1996) Multicolour whole-mount in situ hybridization to Drosophila embryos. Dev Genes Evol 206(4):292–295. doi:10.1007/s004270050055

    Article  CAS  PubMed  Google Scholar 

  19. Hauptmann G, Gerster T (2000) Multicolor whole-mount in situ hybridization. Methods Mol Biol 137:139–148. doi:10.1385/1-59259-066-7:139

    CAS  PubMed  Google Scholar 

  20. Hauptmann G (1999) Two-color detection of mRNA transcript localizations in fish and fly embryos using alkaline phosphatase and beta-galactosidase conjugated antibodies. Dev Genes Evol 209(5):317–321. doi: 10.1007/s004270050258

  21. Lauter G, Söll I, Hauptmann G (2011) Two-color fluorescent in situ hybridization in the embryonic zebrafish brain using differential detection systems. BMC Dev Biol 11:43. doi:10.1186/1471-213X-11-43

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  22. Lauter G, Söll I, Hauptmann G (2014) Sensitive whole-mount fluorescent in situ hybridization in zebrafish using enhanced tyramide signal amplification. Methods Mol Biol 1082:175–185. doi:10.1007/978-1-62703-655-9_12

    Article  PubMed  Google Scholar 

  23. Hauptmann G (2001) One-, two-, and three-color whole-mount in situ hybridization to Drosophila embryos. Methods 23(4):359–372. doi:10.1006/meth.2000.1148

  24. Hauptmann G, Gerster T (2000) Regulatory gene expression patterns reveal transverse and longitudinal subdivisions of the embryonic zebrafish forebrain. Mech Dev 91(1–2):105–118. doi: 10.1016/S0925-4773(99)00277-4

  25. Hauptmann G, Söll I, Gerster T (2002) The early embryonic zebrafish forebrain is subdivided into molecularly distinct transverse and longitudinal domains. Brain Res Bull 57(3–4):371–375. doi: 10.1016/S0361-9230(01)00691-8

  26. Lauter G, Söll I, Hauptmann G (2013) Molecular characterization of prosomeric and intraprosomeric subdivisions of the embryonic zebrafish diencephalon. J Comp Neurol 521(5):1093–1118. doi:10.1002/cne.23221

    Article  CAS  PubMed  Google Scholar 

  27. Lauter G, Söll I, Hauptmann G (2011) Multicolor fluorescent in situ hybridization to define abutting and overlapping gene expression in the embryonic zebrafish brain. Neural Dev 6(1):10. doi:10.1186/1749-8104-6-10

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  28. Hauptmann G, Lauter G, Söll I (2015) Application of alkaline phosphatase-mediated azo dye staining for dual fluorescent in situ hybridization in zebrafish. In: Hauptmann G (ed) In situ hybridization methods. Neuromethods. Humana Press, New York, vol 99 chapter 20. doi: 10.1007/978-1-4939-2303-8_20

    Chapter  Google Scholar 

  29. Söll I, Hauptmann G (2015) Multicolored visualization of transcript distributions in Drosophila embryos. In: Hauptmann G (ed) In situ hybridization methods. Neuromethods. Humana Press, New York, vol 99 chapter 3. doi: 10.1007/978-1-4939-2303-8_3

  30. Hauptmann G, Gerster T (1996) Complex expression of the zp-50 pou gene in the embryonic zebrafish brain is altered by overexpression of sonic hedgehog. Development 122(6):1769–1780

    CAS  PubMed  Google Scholar 

  31. Hauptmann G, Gerster T (2000) Combinatorial expression of zebrafish Brn-1- and Brn-2-related POU genes in the embryonic brain, pronephric primordium, and pharyngeal arches. Dev Dyn 218(2):345–358. doi:10.1002/(SICI)1097-0177(200006)218:2<345::AID-DVDY8>3.0.CO;2-V

    Article  CAS  PubMed  Google Scholar 

  32. Kitambi SS, Hauptmann G (2007) The zebrafish orphan nuclear receptor genes nr2e1 and nr2e3 are expressed in developing eye and forebrain. Gene Expr Patterns 7(4):521–528. doi:10.1016/j.modgep.2006.10.006

  33. Bräutigam L, Hillmer JM, Söll I, Hauptmann G (2010) Localized expression of urocortin genes in the developing zebrafish brain. J Comp Neurol 518(15):2978–2995. doi:10.1002/cne.22375

    Article  PubMed  Google Scholar 

  34. Chandrasekar G, Lauter G, Hauptmann G (2007) Distribution of corticotropin-releasing hormone in the developing zebrafish brain. J Comp Neurol 505(4):337–351. doi:10.1002/cne.21496

    Article  CAS  PubMed  Google Scholar 

  35. Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF (1995) Stages of embryonic development of the zebrafish. Dev Dyn 203(3):253–310. doi:10.1002/aja.1002030302

    Article  CAS  PubMed  Google Scholar 

  36. Plickert G, Gajewski M, Gehrke G, Gausepohl H, Schlossherr J, Ibrahim H (1997) Automated in situ detection (AISD) of biomolecules. Dev Genes Evol 207(5):362–367

    Article  CAS  Google Scholar 

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Acknowledgements

We thank Prof. Dr. Ulrich Theopold and the Wenner Gren Institute of Stockholm University for their support, while preparing this manuscript. We thank Robert Krautz for preparing schematic Figs. 1 and 2. Automated in situ hybridization was established when we were working at Södertörn University.

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Authors and Affiliations

  1. Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, 10691, Stockholm, Sweden

    Iris Söll & Giselbert Hauptmann Ph.D.

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  1. Iris Söll
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  2. Giselbert Hauptmann Ph.D.
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Correspondence to Giselbert Hauptmann Ph.D. .

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  1. Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden

    Giselbert Hauptmann

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Söll, I., Hauptmann, G. (2015). Manual and Automated Whole-Mount In Situ Hybridization for Systematic Gene Expression Analysis in Embryonic Zebrafish Forebrain. In: Hauptmann, G. (eds) In Situ Hybridization Methods. Neuromethods, vol 99. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2303-8_9

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  • DOI: https://doi.org/10.1007/978-1-4939-2303-8_9

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2302-1

  • Online ISBN: 978-1-4939-2303-8

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