Expression Profile And Methylation Status Of Lncrna In Dlk1-dio3Region During Mouse Preimplantation Development

Recent years, long non-coding RNA (lnc RNA) in Dlk1-Dio3gene cluster have gained widespread attention because of their closely relationship with induced pluripotent stem cell (iPS). This study focused on the main lncRNAs (Gtl2, Rian and Mirg) in Dlk1-Dio3gene cluster, and analyzed theirs spatiotemporal expression patterns by in situ hybridization and fluorescence in situ hybridization(FISH). and the methylation status of two important differentially methylated regions (IG-DMR,Gtl2-DMR) were detected by bisulfite genomic sequencing during mouse preimplantation development. Additionally, variety expression of lncRNAs and methylation dynamic of DMRs in TM3treated with methyltransferase inhibitor were also analyzed.Firstly a protocol of whole in situ hybridization based on pore plate and drop was established in this study. In situ hybridization indicated that these lncRNAs only expressed in the morula and blastocyst, while not in8-cell stage and before. Interestingly, the expression of lncRNAs is higher in blastocyst stage than that in morula, which in accordance with the results of real-time RT-PCR. More importantly, we found the expression signals of these lncRNAs were concentrated in the inner cell mass (ICM) of the blastocyst, but almost not in trophectoderm (TE). FISH illustrated that these lncRNAs’ signal mainly surrounding cell nucleus in blastocyst. As analyzed by bisulfite sequencing analysis, from2-cell to morula stage, IG-DMR still maintained its differentially methylated state established in the germ line. That is maternal non-methylated and paternal methylated. Gtl2-DMR showed low methylated state on both maternal and paternal strands throughout the preimplantation development, and appeared no obvious changes. What’s more, the expression of these lncRNAs was not regulated by DNA methylation in TM3.This is the first study to clarify the expression patterns of lncRNAs in Dlk1-Dio3gene cluster during the preimplantation development. The expression of lncRNAs at morula and blastocyst stage, and intense signals from ICM of blastocyst strongly indicated the possible association between these lncRNAs and ESCs. The methylated states of DMRs did not change during the preimplantation development. It seems expressions of these lncRNAs are not controlled by DNA methylation of DMRs, which mainly function likely on maintaining imprinting status of these lncRNAs.