Research On Expression, Regulation, And Function Of LncRNA Gtl2during Pre-implantation Embryonic Development In Mice

The long non-coding RNAs (lncRNA) is a class ofnon-protein-coding RNAmolecules, which participate in the regulation of gene imprinting and embryonicdevelopment. However, there is a lack of relevant research on expression patternsand functions of lncRNA in preimplantation development. Recent studies revealedthe important role of lncRNAs (including Gtl2, Rian and Mirg) from theDlk1-Dio3region on the pluripotency of iPS cells. Transcriptional silencing ofthese maternal expressed genes (MEGs) resulted in the failure to generate all-iPSCmice, and the degree of activation of this region was positively correlated with thepluripotency level of stem cells.As the close relationships between the iPSCs and ES cells, and the ES cellsare derived from inner cell mass(ICM) of blastocyst embryos, we wonder theexpressed patterns and functions of these lncRNAs in preimplantation embryos.Our previous studies have shown that these lncRNAs all start to express at morulaand highest in blastocyst, while the activation of lncRNAs expression is notsubject to the regulation of IG-DMR and Gtl2-DMR DNA methylation.Subject tothe mouse Dlk1-Dio3imprinting cluster model, we will study expression patterns,regulation and functions of the cluster of the most important lncRNA-Gtl2duringearly mouse embryogenesis.Firstly, we will make it clear in expression patterns of lncRNA Gtl2duringpreimplantation development. In order to explore the dynamic expression changesand the subcellular localization from morlua to balstocyst, we carried out RNAFISH in morula, early blastocyst and blastocyst. A clear concentration expressiontrend to inner cells mass was observed accompanied with embryo developmentfrom morula to blastocyst. In blastocyst, lncRNA Gtl2is almost concentrated inthe inner cell mass, hardly detected in the trophoblast cells. lncRNA Rian andMirg have the similar expression pattern. In addition, we analyzed Gtl2imprintedpattern at morula stage, the results showed the imprinted pattern of lncRNA Gtl2was established when it starts to express at morula, that is, maternal expressed. Thespatiotemporal expression patterns and dynamic changes of these lncRNAssuggest their potential crucial functions during early embryo development.Given the interesting spatiotemporal expression patterns and dynamicchanges of lncRNA Gtl2, secondly, we analyzed Gtl2epigenetic regulationmechanism. As our previous work has shown no changes were found in IG-DMRand Gtl2-DMR before and after lncRNA Gtl2expression which suggested itsactivation is not subject to the regulation of two DMRs DNA methylation, so we check the histone modifications in the promoter regions. We choose H3k9me3,H3K27me3, H3K4me3and H3ac to perform the microChIP assay (following up amicro ChIP method published on nature protocols), and found lncRNA expressionlevel is rise along with the increase of H3K4me3at its promoter from8-cell stageto blastocyst.In order to understand Gtl2expression regulation mechanism fromtranscription factor aspect, thirdly, we constructed shRNA lentivirus vectorsrespectively targeted Oct4, Sox2and Nanog, three key transcriptional factors inES cells, and successfully knocked down them by lentiviral particle microinjectionto perivitelline space at zygote period. After culturing in vitro for4days, wecollect these embryos respectively and detected the expression of lncRNA byqRT-PCR. We found each of three transcriptional factors inhibition candownregulate the lncRNA expression. The results suggest stem cell transcriptionalfactorsmay influence the lncRNA expression by direct or indirect way.At last, we studied the functions of lncRNA Gtl2during preimplantationdevelopment. We knocked down lncRNA Gtl2by shRNA lentiviral particlemicroinjection. Although about60%of Gtl2was knocked down, it didn’tinfluence the blastocyst formation rate compared to control group. However,interference of Gtl2compromises both TE and ICM outgrowth potential, and theadjacent gene from Dlk1-Dio3imprinted region and some stem cell pluripotencyfactors are widely downregulated following Gtl2interfered.This study revealed lncRNA Gtl2possessed specific spatiotemporalexpression and location, complicated transcription regulation mechanism andsignificant functions in mouse early embryos, implying Gtl2was a functionallncRNA in embryo development, cell differentiation and regulation of geneexpression.