Dynamic transition of Dnmt3b expression in mouse pre- and early post-implantation embryos

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Abstract

The de novo DNA methyltransferases, Dnmt3a and Dnmt3b, are responsible for the creation of DNA methylation patterns in mouse development. Dnmt3b is more highly expressed in early developmental stages than Dnmt3a, and is thought to have an important role in the epigenetic gene regulation during early embryogenesis. Previous reports suggest that Dnmt3b is expressed preferentially in the embryonic lineage, but less in the extra-embryonic lineage, in early post-implantation embryos. However, it is unclear when this lineage-specific differential expression is established. Here we demonstrate that Dnmt3b shows a dynamic expression change during pre- and early post-implantation development. Contrary to the expectation, Dnmt3b is preferentially expressed in the trophectoderm rather than the inner cell mass at the mid blastocyst stage. Subsequently, the spatial Dnmt3b expression gradually changes during pre- and early post-implantation development, and finally Dnmt3b expression is settled in the embryonic lineage at the epiblast stage. The findings are consistent with the role for Dnmt3b in cell-lineage specification and the creation of lineage-specific DNA methylation patterns.

Section snippets

Results and discussion

In mammalian development, the first cell-lineage-allocation occurs in the morula stage, giving rise to the inner cell mass (ICM, embryonic lineage) and trophectoderm (TE, extra-embryonic lineage) of the blastocyst. Differential gene expression and epigenetic modifications are observed between the two lineages (Morgan et al., 2005). One such differential modification is ICM-specific de novo DNA methylation (Dean et al., 2001, Santos et al., 2002), but it is unknown which DNA methyltransferase is

Preparation of embryo

Embryos were obtained from ICR mice. Preparation of morulae (16- to 32-cell, E3.0) and blastocysts (with a middle sized blastocele, E3.5) was done as described previously (Hirasawa et al., 2008). E4.0 (hatched blastocysts) and E4.5 embryos were flushed from the uteri with PBS. E5.5 post-implantation embryos were dissected from the desidual tissues.

Antibodies

The anti-Dnmt3b monoclonal antibody (IMG-184, IMGENEX) recognized all known active isoforms of Dnmt3b. The specificity of the antibody was tested

Acknowledgements

We thank Drs. E. Li and M. Okano for providing Dnmt3b conditional knockout mice; Drs. M. Kaneda, Y. Kato, T. Sado and K. Hata for helpful discussion; H. Chiba, H. Furuumi and H. Inoue for technical assistance. This work was supported in part by Grant-in-Aid for Scientific Research on Priority Area from the Ministry of Education, Culture, Sports, Science and Technology of Japan to H.S. R.H. thanks the Japanese Society for the Promotion of Science for Young Scientists Fellowship.

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