Title:
See, manipulate, and reconstitute the chromatin and nucleus
Speaker:
Kazuo Yamagata (Faculty of Biology-Oriented Science and Technology, Kindai University)
Abstract:
Fertilization and subsequent preimplantation development are unique processes that result in rapid cell proliferation and simultaneous cellular differentiation. Meanwhile, repeated construction and destruction of the nuclei and chromosome segregations are seen, and inside nuclei, global changes in epigenetic markers take place. To understand their mechanisms and significance, molecular approaches, such as gene disruption, are now widespread. In addition, quantitative and reconstitution approaches would aid understanding the spatiotemporal regulation and relationship of these elements. Therefore, we have developed some methodologies to examine them. One is live-cell imaging in which mRNA-encoding fluorescent probes and/or fluorescently labeled antibodies are injected into an oocyte or zygote, which are examined by spinning-disk confocal microscopy. Chromosome segregations (1), dynamic changes in DNA (2), and histone modifications (3) can be monitored in this way. The main advantage of our imaging is that it has an ultralow invasive character. This characteristic has allowed observations and quantifications over several days from fertilization to the blastocyst stage in murine, bovine (4), and human embryos. The next important methodology is epigenetic manipulation. This method is based on gene-editing technology and we have succeeded in specifically and artificially introducing DNA methylation into the centromere or pericentromeric region in mouse embryos (5, 6). The last method to be discussed is nuclear reconstruction in a living mouse zygote. In this technique, a known DNA fragment is coated with small (about 3 mm diameter) magnetic beads, which are injected into oocytes or zygotes to visualize activity around the beads (7). Using the same methodology, we reported signs of nuclear formation ability of mammoth remains (8). In this seminar, I am going to talk about the details of methodologies mentioned and recent findings using them.
References:
1: Mashiko D, Ikeda Z, Yao T, Tokoro M, Fukunaga N, Asada Y, Yamagata K. Chromosome segregation error during early cleavage in mouse pre-implantation embryo does not necessarily cause developmental failure after blastocyst stage. Sci Rep. 2020 Jan 21;10(1):854.
2: Ueda J, Maehara K, Mashiko D, Ichinose T, Yao T, Hori M, Sato Y, Kimura H, Ohkawa Y, Yamagata K. Heterochromatin dynamics during the differentiation process revealed by the DNA methylation reporter mouse, MethylRO. Stem Cell Reports. 2014 Jun 3;2(6):910-24.
3: Kimura H, Yamagata K. Visualization of epigenetic modifications in preimplantation embryos. Methods Mol Biol. 2015;1222:127-47.
4: Yao T, Suzuki R, Furuta N, Suzuki Y, Kabe K, Tokoro M, Sugawara A, Yajima A, Nagasawa T, Matoba S, Yamagata K, Sugimura S. Live-cell imaging of nuclear-chromosomal dynamics in bovine in vitro fertilised embryos. Sci Rep. 2018 May 10;8(1):7460.
5: Yamazaki T, Hatano Y, Handa T, Kato S, Hoida K, Yamamura R, Fukuyama T, Uematsu T, Kobayashi N, Kimura H, Yamagata K. Targeted DNA methylation in pericentromeres with genome editing-based artificial DNA methyltransferase. PLoS One. 2017 May 18;12(5):e0177764.
6: Yamazaki T, Hatano Y, Taniguchi R, Kobayashi N, Yamagata K. Editing DNA Methylation in Mammalian Embryos. Int J Mol Sci. 2020 Jan 18;21(2). pii: E637.
7: Suzuki Y, Bilir Ş, Hatano Y, Fukuda T, Mashiko D, Kobayashi S, Hiraoka Y, Haraguchi T, Yamagata K. Nuclear formation induced by DNA-conjugated beads in living fertilised mouse egg. Sci Rep. 2019 Jun 11;9(1):8461.
8: Yamagata K, Nagai K, Miyamoto H, Anzai M, Kato H, Miyamoto K, Kurosaka S, Azuma R, Kolodeznikov II, Protopopov AV, Plotnikov VV, Kobayashi H, Kawahara-Miki R, Kono T, Uchida M, Shibata Y, Handa T, Kimura H, Hosoi Y, Mitani T, Matsumoto K, Iritani A. Signs of biological activities of 28,000-year-old mammoth nuclei in mouse oocytes visualized by live-cell imaging. Sci Rep. 2019 Mar 11;9(1):4050.
