Advertisement

Multicolored Visualization of Transcript Distributions in Drosophila Embryos

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

Abstract

Despite large-scale gene expression profiling studies, it is still often required to precisely characterize the localization of different transcripts in relation to each other to determine unique and overlapping expression sites at a cellular level. We describe here a versatile protocol for simultaneous examination of three unique mRNA expression patterns in Drosophila melanogaster embryos. Three differently labeled antisense RNA probes are hybridized together to the embryos and detected by sequential alkaline phosphatase-based immunohistochemistry. Transcript distributions are revealed by colorimetric enzymatic reactions that permit to highlight each mRNA expression pattern by a differing and contrasting cellular color precipitate. We provide tips and tricks for each critical step helpful for successful application of the tricolor whole-mount in situ hybridization (WISH) method.

Key words

Digoxigenin Biotin Fluorescein Azo dye Fast Blue Fast Red INT Alkaline phosphatase substrate Gene expression analysis RNA localization 

Notes

Acknowledgements

We thank Prof. Dr. Ulrich Theopold and the Wenner Gren Institute of Stockholm University for their support, while preparing this manuscript. This method was initially developed during G.H.’s stay in the laboratory of Dr. Thomas Gerster at the Biozentrum of the Universität Basel.

References

  1. 1.
    Tautz D, Pfeifle C (1989) A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback. Chromosoma 98(2):81–85CrossRefPubMedGoogle Scholar
  2. 2.
    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 CrossRefPubMedGoogle Scholar
  3. 3.
    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 CrossRefPubMedGoogle Scholar
  4. 4.
    Hartmann C, Jäckle H (1995) Spatiotemporal relationships between a novel Drosophila stripe expressing gene and known segmentation genes by simultaneous visualization of transcript patterns. Chromosoma 104(2):84–91CrossRefPubMedGoogle Scholar
  5. 5.
    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 CrossRefPubMedGoogle Scholar
  6. 6.
    O’Neill JW, Bier E (1994) Double-label in situ hybridization using biotin and digoxigenin-tagged RNA probes. Biotechniques 17(5):870, 874–875PubMedGoogle Scholar
  7. 7.
    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 CrossRefPubMedGoogle Scholar
  8. 8.
    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 CrossRefPubMedGoogle Scholar
  9. 9.
    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 CrossRefPubMedGoogle Scholar
  10. 10.
    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 PubMedGoogle Scholar
  11. 11.
    Hauptmann G, Belting HG, Wolke U, Lunde K, Söll I, Abdelilah-Seyfried S, Prince V, Driever W (2002) spiel ohne grenzen/pou2 is required for zebrafish hindbrain segmentation. Development 129(7):1645–1655PubMedGoogle Scholar
  12. 12.
    Hauptmann G, Gerster T (1995) Pou-2—a zebrafish gene active during cleavage stages and in the early hindbrain. Mech Dev 51(1):127–138, doi: 10.1016/0925-4773(95)00360-D CrossRefPubMedGoogle Scholar
  13. 13.
    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):1769–1780PubMedGoogle Scholar
  14. 14.
    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 CrossRefPubMedGoogle Scholar
  15. 15.
    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 CrossRefPubMedGoogle Scholar
  16. 16.
    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):362–367CrossRefGoogle Scholar
  17. 17.
    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 (ed) In situ hybridization methods. Neuromethods. Humana, New York, vol 99 chapt 9, doi: 10.1007/978-1-4939-2303-8_9
  18. 18.
    Wolff C, Sommer R, Schröder R, Glaser G, Tautz D (1995) Conserved and divergent expression aspects of the Drosophila segmentation gene hunchback in the short germ band embryo of the flour beetle Tribolium. Development 121(12):4227–4236PubMedGoogle Scholar
  19. 19.
    Wolff C, Schröder R, Schulz C, Tautz D, Klingler M (1998) Regulation of the Tribolium homologues of caudal and hunchback in Drosophila: evidence for maternal gradient systems in a short germ embryo. Development 125(18):3645–3654PubMedGoogle Scholar
  20. 20.
    Chang CC, Huang TY, Shih CL, Lin GW, Chang TP, Chiu H, Chang WC (2008) Whole-mount identification of gene transcripts in aphids: protocols and evaluation of probe accessibility. Arch Insect Biochem Physiol 68(4):186–196CrossRefPubMedGoogle Scholar
  21. 21.
    Hansen GN, Williamson M, Grimmelikhuijzen CJ (2000) Two-color double-labeling in situ hybridization of whole-mount Hydra using RNA probes for five different Hydra neuropeptide preprohormones: evidence for colocalization. Cell Tissue Res 301(2):245–253CrossRefPubMedGoogle Scholar
  22. 22.
    Mitgutsch C, Hauser F, Grimmelikhuijzen CJ (1999) Expression and developmental regulation of the Hydra-RFamide and Hydra-LWamide preprohormone genes in Hydra: evidence for transient phases of head formation. Dev Biol 207(1):189–203CrossRefPubMedGoogle Scholar
  23. 23.
    Oliveri P, Davidson EH, McClay DR (2003) Activation of pmar1 controls specification of micromeres in the sea urchin embryo. Dev Biol 258(1):32–43CrossRefPubMedGoogle Scholar
  24. 24.
    Wada H, Holland PW, Sato S, Yamamoto H, Satoh N (1997) Neural tube is partially dorsalized by overexpression of HrPax-37: the ascidian homologue of Pax-3 and Pax-7. Dev Biol 187(2):240–252CrossRefPubMedGoogle Scholar
  25. 25.
    Hurtado R, Mikawa T (2006) Enhanced sensitivity and stability in two-color in situ hybridization by means of a novel chromagenic substrate combination. Dev Dyn 235(10):2811–2816CrossRefPubMedGoogle Scholar
  26. 26.
    Monuki ES, Porter FD, Walsh CA (2001) Patterning of the dorsal telencephalon and cerebral cortex by a roof plate-Lhx2 pathway. Neuron 32(4):591–604CrossRefPubMedGoogle Scholar
  27. 27.
    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 CrossRefPubMedCentralPubMedGoogle Scholar
  28. 28.
    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 CrossRefPubMedGoogle Scholar
  29. 29.
    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, New York, vol 99 chapt 20, doi: 10.1007/978-1-4939-2303-8_20
  30. 30.
    Legendre F, Cody N, Iampietro C, Bergalet J, Lefebvre FA, Moquin-Beaudry G, Zhang O, Wang X, Lécuyer E (2013) Whole mount RNA fluorescent in situ hybridization of Drosophila embryos. J Vis Exp 71:e50057PubMedGoogle Scholar
  31. 31.
    Bergalet J, Iampietro C, Chin A, Nguyen X-T, Ore-Rodriguez S, Cody N, Lecuyer E (2015) Subcellular transcript localization in Drosophila embryos and tissues visualized by multiplex FISH. In: Hauptmann G (ed) In situ hybridization methods. Neuromethods. Humana, New York, vol 99 chapt 19, doi: 10.1007/978-1-4939-2303-8_19
  32. 32.
    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 CrossRefPubMedCentralPubMedGoogle Scholar
  33. 33.
    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 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of Molecular Biosciences, The Wenner-Gren InstituteStockholm UniversityStockholmSweden

Personalised recommendations