Abstract
Preimplantation development is a complicated process, which involves many genes. We have investigated the expression patterns of 17 developmentally important genes and isoforms in early mouse embryos as well as in single cells of the mouse embryo. The comparison is an excellent example for showing the importance of studying heterogeneity among cell populations on the RNA level, which is being increasingly addressed in basic research and medical sciences, particularly with a link to diagnostics (e.g. the analysis of circulating tumor cells and their progenitors). The ubiquitously expressed histone variant H3f3a and the transcription factor Pou5f1 generated mRNA-derived products in all analyzed preimplantation embryos (up to the morula stage) and in all analyzed blastomeres from 16-cell embryos, indicating a rather uniform reactivation of pluripotency gene expression during mouse preimplantation development. In contrast, genes that have been implicated in epigenetic genome reprogramming, such as DNA methyltransferases, methylcytosine-binding proteins, or base excision repair genes revealed considerable variation between individual cells from the same embryo and even higher variability between cells from different embryos. We conclude that at a given point of time, the transcriptome encoding the reprogramming machinery and, by extrapolation, genome reprogramming differs between blastomeres. It is tempting to speculate that cells expressing the reprogramming machinery have a higher developmental potential.
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References
Levsky JM, Singer RH (2003) Gene expression and the myth of the average cell. Trends Cell Biol 13:4-6
Raj A, Peskin CS, Tranchina D, Vargas DY, Tyagi S (2006) Stochastic mRNA synthesis in mammalian cells. PLOS Biol 4:e309
Bestor TH (2000) The DNA methyltransferases of mammals. Hum Mol Genet 9:2395-2402
Li E, Bestor TH, Jaenisch R (1992) Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 69:915-926
Okano M, Bell DW, Haber DA, Li E (1999) DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 99:247-257
Aoki A, Suetake I, Miyagawa J, Fujio T, Chijiwa T, Sasaki H, Tajima S (2001) Enzymatic properties of de novo-type mouse DNA (cytosine-5) methyltransferases. Nucl Acids Res 29:506-512
Chen T, Ueda Y, Dodge JE, Wang Z, Li E (2003) Establishment and maintenance of genomic methylation patterns in mouse embryonic stem cells by Dnmt3a and Dnmt3b. Mol Cell Biol 23:5594-5605
Suetake I, Shinozaki F, Miyagawa J, Takeshima H, Tajima S (2004) DNMT3L stimulates the DNA methylation activity of Dnmt3a and Dnmt3b through a direct interaction. J Biol Chem 279:27816-27823
Ballestar E, Wolffe AP (2001) Methyl-CpG-binding proteins. Targeting specific gene repression. Eur J Biochem 268:1-6
Jaenisch R, Bird A (2003) Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nat Genet 33:245-254
Bhattacharya SK, Ramchandani S, Cervoni N, Szyf M (1999) A mammalian protein with specific demethylase activity for mCpG DNA. Nature 397:579-583
Jost JP (1993) Nuclear extracts of chicken embryos promote an active demethylation of DNA by excision repair of 5-methyldeoxycytidine. Proc Natl Acad Sci USA 90:4684-4688
Evans AR, Limp-Foster M, Kelley MR (2000) Going APE over ref-1. Mutat Res 461:83-108
Sancar A, Lindsey-Boltz LA, Unsal-Kacmaz K, Linn S (2004) Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints. Annu Rev Biochem 73:39-85
Wilmut I, Beaujean N, de Sousa PA, Dinnyes A, King TJ, Paterson LA, Wells DN, Young LE (2002) Somatic cell nuclear transfer. Nature 419:583-586
Schulz RM (1993) Regulation of zygotic gene activation in the mouse. BioEssays 8:531-538
Palmieri SL, Peter W, Hess H, Schöler HR (1994) OCT-4 transcription factor is differentially expressed in the mouse embryo during establishment of the first two extraembryonic cell lineages involved in implantation. Dev Biol 166:259-267
Nichols J, Zevnik B, Anastassiadis K, Niwa H, Klewe-Nebenius D, Chambers I, Schöler HR, Smith A (1998) Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell 95:379-391
Torres-Padilla ME, Bannister AJ, Hurd PJ, Kouzarides T, Zernicka-Goetz M (2006) Dynamic distribution of the replacement histone variant H3.3 in the mouse oocyte and preimplantation embryos. Int J Dev Biol 50:455-461
May A, Kirchner R, Müller H, Hartmann P, El Hajj N, Tresch A, Zechner U, Mann W, Haaf T (2009) Multiplex RT-PCR expression analysis of developmentally important genes in individual mouse preimplantation embryos and blastomeres. Biol Reprod 80:194-202. doi:10.1095/biolreprod.107.064691
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Mann, W., Haaf, T. (2010). Single Cell RT-PCR on Mouse Embryos: A General Approach for Developmental Biology. In: King, N. (eds) RT-PCR Protocols. Methods in Molecular Biology, vol 630. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-629-0_1
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DOI: https://doi.org/10.1007/978-1-60761-629-0_1
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