Abstract
RNA in situ hybridization is a practical technique that allows investigators to observe temporal and spatial gene expression at the RNA level in the context of whole embryos or tissues. One powerful application of in situ hybridization is to observe the consequences of genetic, toxicologic, or environmental perturbations on gene expression or morphogenesis during development. Herein, I will review the procedure to perform nonradioactive, in situ hybridization on whole-mount mouse or chick embryos.
Key words
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Gall JG, Pardue ML (1969) Formation and detection of RNA-DNA hybrid molecules in cytological preparations. Proc Natl Acad Sci U S A 63:378–383
Acloque H, Wilkinson DG, Nieto MA (2008) In situ hybridization analysis of chick embryos in whole-mount and tissue sections. Methods Cell Biol 87:169–185
Ortega FG, Lorente JA, Garcia Puche JL, Ruiz MP, Sanchez-Martin RM, de Miguel-Perez D, Diaz-Mochon JJ, Serrano MJ (2015) miRNA in situ hybridization in circulating tumor cells—MishCTC. Sci Rep 5:9207
Swennenhuis JF, Tibbe AG, Levink R, Sipkema RC, Terstappen LW (2009) Characterization of circulating tumor cells by fluorescence in situ hybridization. Cytometry A 75:520–527
Wilkinson DG, Nieto MA (1993) Detection of messenger RNA by in situ hybridization to tissue sections and whole mounts. Methods Enzymol 225:361–373
Wang J, Hamblet NS, Mark S, Dickinson ME, Brinkman BC, Segil N, Fraser SE, Chen P, Wallingford JB, Wynshaw-Boris A (2006) Dishevelled genes mediate a conserved mammalian PCP pathway to regulate convergent extension during neurulation. Development 133:1767–1778
Crossley PH, Martin GR (1995) The mouse Fgf8 gene encodes a family of polypeptides and is expressed in regions that direct outgrowth and patterning in the developing embryo. Development 121:439–451
Crossley PH, Minowada G, MacArthur CA, Martin GR (1996) Roles for FGF8 in the induction, initiation, and maintenance of chick limb development. Cell 84:127–136
Briscoe J, Sussel L, Serup P, Hartigan-O’Connor D, Jessell TM, Rubenstein JL, Ericson J (1999) Homeobox gene Nkx2.2 and specification of neuronal identity by graded Sonic hedgehog signalling. Nature 398:622–627
Takada S, Stark KL, Shea MJ, Vassileva G, McMahon JA, McMahon AP (1994) Wnt-3a regulates somite and tailbud formation in the mouse embryo. Genes Dev 8:174–189
Yamaguchi TP, Takada S, Yoshikawa Y, Wu N, McMahon AP (1999) T (Brachyury) is a direct target of Wnt3a during paraxial mesoderm specification. Genes Dev 13:3185–3190
Kohn AD, Moon RT (2005) Wnt and calcium signaling: beta-catenin-independent pathways. Cell Calcium 38:439–446
Bel-Vialar S, Core N, Terranova R, Goudot V, Boned A, Djabali M (2000) Altered retinoic acid sensitivity and temporal expression of Hox genes in polycomb-M33-deficient mice. Dev Biol 224:238–249
Bel-Vialar S, Itasaki N, Krumlauf R (2002) Initiating Hox gene expression: in the early chick neural tube differential sensitivity to FGF and RA signaling subdivides the HoxB genes in two distinct groups. Development 129:5103–5115
Ho NY, Yang L, Legradi J, Armant O, Takamiya M, Rastegar S, Strahle U (2013) Gene responses in the central nervous system of zebrafish embryos exposed to the neurotoxicant methyl mercury. Environ Sci Technol 47:3316–3325
Hong M, Krauss RS (2017) Ethanol itself is a holoprosencephaly-inducing teratogen. PLoS One 12:e0176440
Huyck RW, Nagarkar M, Olsen N, Clamons SE, Saha MS (2015) Methylmercury exposure during early Xenopus laevis development affects cell proliferation and death but not neural progenitor specification. Neurotoxicol Teratol 47:102–113
Kot-Leibovich H, Fainsod A (2009) Ethanol induces embryonic malformations by competing for retinaldehyde dehydrogenase activity during vertebrate gastrulation. Dis Model Mech 2:295–305
Lee LM, Leung CY, Tang WW, Choi HL, Leung YC, McCaffery PJ, Wang CC, Woolf AS, Shum AS (2012) A paradoxical teratogenic mechanism for retinoic acid. Proc Natl Acad Sci U S A 109:13668–13673
Lee LM, Leung MB, Kwok RC, Leung YC, Wang CC, McCaffery PJ, Copp AJ, Shum AS (2017) Perturbation of retinoid homeostasis increases malformation risk in embryos exposed to pregestational diabetes. Diabetes 66:1041–1051
Marshall H, Morrison A, Studer M, Popperl H, Krumlauf R (1996) Retinoids and Hox genes. FASEB J 10:969–978
Soderstrom S, Ebendal T (1995) In vitro toxicity of methyl mercury: effects on nerve growth factor (NGF)-responsive neurons and on NGF synthesis in fibroblasts. Toxicol Lett 75:133–144
Luehrsen KR, Davidson S, Lee YJ, Rouhani R, Soleimani A, Raich T, Cain CA, Collarini EJ, Yamanishi DT, Pearson J et al (2000) High-density hapten labeling and HRP conjugation of oligonucleotides for use as in situ hybridization probes to detect mRNA targets in cells and tissues. J Histochem Cytochem 48:133–145
Nagy A, Gertenstein M, Vintersten K, Behringer R (2003) Manipulating the mouse embryo: a laboratory manual, 3rd edn. Cold Spring Harbor Press, New York
Hamburger V, Hamilton HL (1992) A series of normal stages in the development of the chick embryo. 1951. Dev Dyn 195:231–272
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Barrow, J.R. (2019). Examining Gene Expression Patterns Through Whole-Mount In Situ Hybridization. In: Hansen, J., Winn, L. (eds) Developmental Toxicology. Methods in Molecular Biology, vol 1965. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9182-2_19
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9182-2_19
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-9181-5
Online ISBN: 978-1-4939-9182-2
eBook Packages: Springer Protocols