Abstract
Immunofluorescence and RNA fluorescence in situ hybridization (FISH) methods enable the detection of, respectively, proteins and RNA molecules in single cells. Adapted to preimplantation mouse embryos, these techniques allow the investigation of transcriptional dynamics in the first embryonic and extraembryonic lineages and can circumvent the limited amount of starting material. This can as well be coupled to examination of chromatin modification, i.e., histone marks, by immunofluorescence. Here is outlined an immunofluorescence protocol combined to nascent RNA-FISH after immunosurgery of the mouse inner cell mass of the blastocyst to study early changes in transcription and/or histone marks of both primitive endoderm and epiblast cells. The method describes the different steps from coverslips and FISH probe preparation to inner cell mass isolation and immunofluorescence followed by RNA-FISH. Furthermore, this is applicable to earlier developmental stages and other mammalian species provided little technical adjustments.
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References
Chazaud C, Yamanaka Y (2016) Lineage specification in the mouse preimplantation embryo. Development 143:1063–1074. https://doi.org/10.1242/dev.128314
Boroviak T, Loos R, Lombard P et al (2015) Lineage-specific profiling delineates the emergence and progression of naive pluripotency in mammalian embryogenesis. Dev Cell 35:366–382. https://doi.org/10.1016/j.devcel.2015.10.011
Surani MA, Hayashi K, Hajkova P (2007) Genetic and epigenetic regulators of pluripotency. Cell 128:747–762. https://doi.org/10.1016/j.cell.2007.02.010
Borensztein M, Okamoto I, Syx L et al (2017) Contribution of epigenetic landscapes and transcription factors to X-chromosome reactivation in the inner cell mass. Nat Commun 8:1–14. https://doi.org/10.1038/s41467-017-01415-5
Okamoto I, Otte AP, Allis CD et al (2004) Epigenetic dynamics of imprinted X inactivation during early mouse development. Science 303:644–649. https://doi.org/10.1126/science.1092727
Chazaud C, Yamanaka Y, Pawson T, Rossant J (2006) Early lineage segregation between epiblast and primitive endoderm in mouse blastocysts through the Grb2-MAPK pathway. Dev Cell 10:615–624. https://doi.org/10.1016/j.devcel.2006.02.020
Solter D, Knowles BB (1975) Immunosurgery of mouse blastocyst. Proc Natl Acad Sci U S A 72:5099–5102. https://doi.org/10.1073/pnas.72.12.5099
Nagy A, Gertsenstein M, Vintersten K, Behringer R (2003) Manipulating the mouse embryo: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
Okamoto I (2018) Combined immunofluorescence, RNA FISH, and DNA FISH in preimplantation mouse embryos. Methods Mol Biol 1861:149–159. https://doi.org/10.1007/978-1-4939-8766-5_12
Ancelin K, Syx L, Borensztein M et al (2016) Maternal LSD1/KDM1A is an essential regulator of chromatin and transcription landscapes during zygotic genome activation. Elife 5:e08851. https://doi.org/10.7554/eLife.08851
Ranisavljevic N, Okamoto I, Heard E, Ancelin K (2017) RNA FISH to study zygotic genome activation in early mouse embryos. Methods Mol Biol 1605:133–145. https://doi.org/10.1007/978-1-4939-6988-3
Acknowledgments
I am grateful to Prof Edith Heard for her constant support and critical input. I thank Dr. Ikuhiro Okamoto, Dr. Katia Ancelin, and Patricia Diabangouaya for their experimental assistance and advices, respectively, in ICM immunosurgery, IF combined to RNA-FISH , and RNA-FISH on preimplantation embryos.
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Borensztein, M. (2021). Investigating the Inner Cell Mass of the Mouse Blastocyst by Combined Immunofluorescence Staining and RNA Fluorescence In Situ Hybridization. In: Ancelin, K., Borensztein, M. (eds) Epigenetic Reprogramming During Mouse Embryogenesis. Methods in Molecular Biology, vol 2214. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0958-3_11
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DOI: https://doi.org/10.1007/978-1-0716-0958-3_11
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