Two-color fluorescent in situ hybridization in the embryonic zebrafish brain using differential detection systems

BMC Developmental Biology - 2011
Gilbert Lauter1, Iris Söll1, Giselbert Hauptmann1
1Department of Biosciences and Nutrition, Karolinska Institutet, SE-141 83, Huddinge, Sweden

Tóm tắt

Abstract Background Whole-mount in situ hybridization (WISH) is extensively used to characterize gene expression patterns in developing and adult brain and other tissues. To obtain an idea whether a novel gene might be involved in specification of a distinct brain subdivision, nucleus or neuronal lineage, it is often useful to correlate its expression with that of a known regional or neuronal marker gene. Two-color fluorescent in situ hybridization (FISH) can be used to compare different transcript distributions at cellular resolution. Conventional two-color FISH protocols require two separate rounds of horseradish peroxidase (POD)-based transcript detection, which involves tyramide signal amplification (TSA) and inactivation of the first applied antibody-enzyme conjugate before the second detection round. Results We show here that the alkaline phosphatase (AP) substrates Fast Red and Fast Blue can be used for chromogenic as well as fluorescent visualization of transcripts. To achieve high signal intensities we optimized embryo permeabilization properties by hydrogen peroxide treatment and hybridization conditions by application of the viscosity-increasing polymer dextran sulfate. The obtained signal enhancement allowed us to develop a sensitive two-color FISH protocol by combining AP and POD reporter systems. We show that the combination of AP-Fast Blue and POD-TSA-carboxyfluorescein (FAM) detection provides a powerful tool for simultaneous fluorescent visualization of two different transcripts in the zebrafish brain. The application of different detection systems allowed for a one-step antibody detection procedure for visualization of transcripts, which significantly reduced working steps and hands-on time shortening the protocol by one day. Inactivation of the first applied reporter enzyme became unnecessary, so that false-positive detection of co-localization by insufficient inactivation, a problem of conventional two-color FISH, could be eliminated. Conclusion Since POD activity is rather quickly quenched by substrate excess, less abundant transcripts can often not be efficiently visualized even when applying TSA. The use of AP-Fast Blue fluorescent detection may provide a helpful alternative for fluorescent transcript visualization, as the AP reaction can proceed for extended times with a high signal-to-noise ratio. Our protocol thus provides a novel alternative for comparison of two different gene expression patterns in the embryonic zebrafish brain at a cellular level. The principles of our method were developed for use in zebrafish but may be easily included in whole-mount FISH protocols of other model organisms.

Từ khóa


Tài liệu tham khảo

Hafen E, Levine M, Garber RL, Gehring WJ: An improved in situ hybridization method for the detection of cellular RNAs in Drosophila tissue sections and its application for localizing transcripts of the homeotic Antennapedia gene complex. EMBO J. 1983, 2: 617-23.

Tautz D, Pfeifle C: 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. 1989, 98: 81-5. 10.1007/BF00291041.

Hauptmann G: Two-color detection of mRNA transcript localizations in fish and fly embryos using alkaline phosphatase and beta-galactosidase conjugated antibodies. Dev Genes Evol. 1999, 209: 317-21. 10.1007/s004270050258.

Hauptmann G: One-, two-, and three-color whole-mount in situ hybridization to Drosophila embryos. Methods. 2001, 23: 359-72. 10.1006/meth.2000.1148.

Hauptmann G, Gerster T: Multicolor whole-mount in situ hybridization. Methods Mol Biol. 2000, 137: 139-48.

Jowett T: Double in situ hybridization techniques in zebrafish. Methods. 2001, 23: 345-58. 10.1006/meth.2000.1147.

O'Neill JW, Bier E: Double-label in situ hybridization using biotin and digoxigenin-tagged RNA probes. Biotechniques. 1994, 17: 870, 874-5.

Hauptmann G, Gerster T: Two-color whole-mount in situ hybridization to vertebrate and Drosophila embryos. Trends Genet. 1994, 10: 266-

Jowett T, Lettice L: Whole-mount in situ hybridizations on zebrafish embryos using a mixture of digoxigenin- and fluorescein-labelled probes. Trends Genet. 1994, 10: 73-4. 10.1016/0168-9525(94)90220-8.

Hauptmann G, Gerster T: Regulatory gene expression patterns reveal transverse and longitudinal subdivisions of the embryonic zebrafish forebrain. Mech Dev. 2000, 91: 105-18. 10.1016/S0925-4773(99)00277-4.

Hauptmann G, Söll I, Gerster T: The early embryonic zebrafish forebrain is subdivided into molecularly distinct transverse and longitudinal domains. Brain Res Bull. 2002, 57: 371-5. 10.1016/S0361-9230(01)00691-8.

Hauptmann G, Belting HG, Wolke U, Lunde K, Söll I, Abdelilah-Seyfried S, Prince V, Driever W: spiel ohne grenzen/pou2 is required for zebrafish hindbrain segmentation. Development. 2002, 129: 1645-55.

Chandrasekar G, Lauter G, Hauptmann G: Distribution of corticotropin-releasing hormone in the developing zebrafish brain. J Comp Neurol. 2007, 505: 337-51. 10.1002/cne.21496.

Bräutigam L, Hillmer JM, Söll I, Hauptmann G: Localized expression of urocortin genes in the developing zebrafish brain. J Comp Neurol. 2010, 518: 2978-95. 10.1002/cne.22375.

Murdoch A, Jenkinson EJ, Johnson GD, Owen JJ: Alkaline phosphatase-fast red, a new fluorescent label. Application in double labelling for cell cycle analysis. J Immunol Methods. 1990, 132: 45-9. 10.1016/0022-1759(90)90396-D.

Larison KD, BreMiller R, Wells KS, Clements I, Haugland RP: Use of a new fluorogenic phosphatase substrate in immunohistochemical applications. J Histochem Cytochem. 1995, 43: 77-83. 10.1177/43.1.7822768.

Bueno D, Skinner J, Abud H, Heath JK: Double in situ hybridization on mouse embryos for detection of overlapping regions of gene expression. Trends Genet. 1996, 12: 385-7. 10.1016/S0168-9525(96)90090-6.

Jowett T, Yan YL: Double fluorescent in situ hybridization to zebrafish embryos. Trends Genet. 1996, 12: 387-9. 10.1016/S0168-9525(96)90091-8.

Bobrow MN, Harris TD, Shaughnessy KJ, Litt GJ: Catalyzed reporter deposition, a novel method of signal amplification. Application to immunoassays. J Immunol Methods. 1989, 125: 279-85. 10.1016/0022-1759(89)90104-X.

Clay H, Ramakrishnan L: Multiplex fluorescent in situ hybridization in zebrafish embryos using tyramide signal amplification. Zebrafish. 2005, 2: 105-11. 10.1089/zeb.2005.2.105.

Denkers N, Garcia-Villalba P, Rodesch CK, Nielson KR, Mauch TJ: FISHing for chick genes: Triple-label whole-mount fluorescence in situ hybridization detects simultaneous and overlapping gene expression in avian embryos. Dev Dyn. 2004, 229: 651-7. 10.1002/dvdy.20005.

Kosman D, Mizutani CM, Lemons D, Cox WG, McGinnis W, Bier E: Multiplex detection of RNA expression in Drosophila embryos. Science. 2004, 305: 846-10.1126/science.1099247.

Tessmar-Raible K, Steinmetz PR, Snyman H, Hassel M, Arendt D: Fluorescent two-color whole mount in situ hybridization in Platynereis dumerilii (Polychaeta, Annelida), an emerging marine molecular model for evolution and development. Biotechniques. 2005, 39: 460, 462, 464-

Lecuyer E, Parthasarathy N, Krause HM: Fluorescent in situ hybridization protocols in Drosophila embryos and tissues. Methods Mol Biol. 2008, 420: 289-302. 10.1007/978-1-59745-583-1_18.

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

Hauptmann G, Gerster T: Multicolour whole-mount in situ hybridization to Drosophila embryos. Development Genes and Evolution. 1996, 206: 292-295. 10.1007/s004270050055.

van Gijlswijk RP, Wiegant J, Raap AK, Tanke HJ: Improved localization of fluorescent tyramides for fluorescence in situ hybridization using dextran sulfate and polyvinyl alcohol. J Histochem Cytochem. 1996, 44: 389-92. 10.1177/44.4.8601698.

Minton AP: Implications of macromolecular crowding for protein assembly. Curr Opin Struct Biol. 2000, 10: 34-9. 10.1016/S0959-440X(99)00045-7.

Hauptmann G, Gerster T: Combinatorial expression of zebrafish Brn-1- and Brn-2-related POU genes in the embryonic brain, pronephric primordium, and pharyngeal arches. Dev Dyn. 2000, 218: 345-58. 10.1002/(SICI)1097-0177(200006)218:2<345::AID-DVDY8>3.0.CO;2-V.

Bolte S, Cordelieres FP: A guided tour into subcellular colocalization analysis in light microscopy. J Microsc. 2006, 224: 213-32. 10.1111/j.1365-2818.2006.01706.x.

Puelles L, Rubenstein JL: Expression patterns of homeobox and other putative regulatory genes in the embryonic mouse forebrain suggest a neuromeric organization. Trends Neurosci. 1993, 16: 472-9. 10.1016/0166-2236(93)90080-6.

Puelles L, Rubenstein JL: Forebrain gene expression domains and the evolving prosomeric model. Trends Neurosci. 2003, 26: 469-76. 10.1016/S0166-2236(03)00234-0.

Wullimann MF, Puelles L: Postembryonic neural proliferation in the zebrafish forebrain and its relationship to prosomeric domains. Anat Embryol (Berl). 1999, 199: 329-48. 10.1007/s004290050232.

Qiu M, Shimamura K, Sussel L, Chen S, Rubenstein JL: Control of anteroposterior and dorsoventral domains of Nkx-6.1 gene expression relative to other Nkx genes during vertebrate CNS development. Mech Dev. 1998, 72: 77-88. 10.1016/S0925-4773(98)00018-5.

Choi HM, Chang JY, Trinh le A, Padilla JE, Fraser SE, Pierce NA: Programmable in situ amplification for multiplexed imaging of mRNA expression. Nat Biotechnol. 2010, 28: 1208-12. 10.1038/nbt.1692.

Kloosterman WP, Wienholds E, de Bruijn E, Kauppinen S, Plasterk RH: In situ detection of miRNAs in animal embryos using LNA-modified oligonucleotide probes. Nat Methods. 2006, 3: 27-9. 10.1038/nmeth843.

Serluca FC, Fishman MC: Pre-pattern in the pronephric kidney field of zebrafish. Development. 2001, 128: 2233-41.

Krauss S, Concordet JP, Ingham PW: A functionally conserved homolog of the Drosophila segment polarity gene hh is expressed in tissues with polarizing activity in zebrafish embryos. Cell. 1993, 75: 1431-44. 10.1016/0092-8674(93)90628-4.

Püschel AW, Gruss P, Westerfield M: Sequence and expression pattern of pax-6 are highly conserved between zebrafish and mice. Development. 1992, 114: 643-51.

Cheesman SE, Layden MJ, Von Ohlen T, Doe CQ, Eisen JS: Zebrafish and fly Nkx6 proteins have similar CNS expression patterns and regulate motoneuron formation. Development. 2004, 131: 5221-32. 10.1242/dev.01397.

Long S, Rebagliati M: Sensitive two-color whole-mount in situ hybridizations using digoxygenin- and dinitrophenol-labeled RNA probes. Biotechniques. 2002, 32: 494-500.

Hopman AH, Ramaekers FC, Speel EJ: Rapid synthesis of biotin-, digoxigenin-, trinitrophenyl-, and fluorochrome-labeled tyramides and their application for In situ hybridization using CARD amplification. J Histochem Cytochem. 1998, 46: 771-7. 10.1177/002215549804600611.

Thông tin
Thông tin xuất bản

BMC Developmental Biology

Thông tin tác giả