The Asymmetric Distribution And Function Of MRNA And 16S MtrRNA In Mouse Preimplantation Embryo

In many organisms,the fate of cells is determined from the time they are 'born'because they inherit cytoplasmic factors that are spatially localized in the oocyte or in the fertilized egg.Such cytoplasmic factors function as determinants when they guarantee specific cell fate.However,the development of the mammalian embryo is different:there was no such asymmetricly distributed determinants and it is regulative.The evidence for this comes from experiments in which the mouse embryo is reorganized by changing the number of cells or the arrangement,after which it can recover and complete development.Whether the principle of prepattrning model was abandoned in the mammalian embryo development is a question puzzled researchers for several decades.In the mouse embryo,the first differences between cells that result in distinct cell lineages have long been thought to arise only as a consequence of differential cell positioning at relatively late preimplantation stages.The cells were all identical before compaction taking place at the 8-cell stage.However,recent findings show that cells in the mouse embryo can differ in their developmental fate and potency as early as the four-cell stage.Levels of H3 methylation at specific arginine residues are different in four-cell blastomeres.Cells with maximal H3 arginine methylation will contribute to the inner cell mass(ICM).Coincidentally,the accessibility of the DNA binding sites for Oct4 is also different in four blastomeres of 4-cell and it can predict cell lineages of the early embryo.After blocking Oct4 access to DNA,cells underwent only symmetric divisions and contributed all their progeny to the extra-embryonic lineage.Lineage tracing studies suggested that the embryonic and abembryonic parts of the mouse blastocyst become separated already by the first cleavage division.But there was no molecular asymmetry between sisiter cells within 2-cell embryos.In order to find out whether there was any difference between the two sister blastomeres on transcriptome level,we screened 272 1-cell overexpressed genes at early 2-cell stage and 131 2-cell overexpressed genes at middle 2-cell stage by whole-mount in situ hybridization(WM-ISH).We didn't find any asymmetric distribution among these 279 genes we have screened,suggesting mouse preimplantation embryo is not like Drosophila embryo in which most mRNAs were asymmetricly distributed.But there maybe some asymmetrically distributed genes not included in our screening,so single cell transcriptome microarray experiments were performed to compare transcriptome profiles of paired sister blastomeres of 2-cell embryos.Paired profiles duplicates show that 645 genes differed between two sister blastomeres among 21786 detected genes.We confirmed genes with 8 fold changes by qRT-PCR with 15 pairs of sister blastomeres.Among 23 genes we examined,15 genes differed in at least 11 pairs of sister blastomeres.It has been reported that both 12S and 16S mitochondrial rRNAs were predominantly distributed in the animal hemisphere of the mature oocyte after examined by WM-ISH.In our studies,we found that 16S mitochondrial ribosome RNA was asymmetrically distributed in mouse embryos at all stages.The amount of mitochondrial ribosome RNA was different between the two blastomeres at 2-cell stage by whole-mount in situ hybridization.This difference also exits in sister blastomeres of 4-cell embryo.At 4-cell and 8-cell stages,the mitochondrial ribosomal RNA is localized basally along the cell-cell junctions.The subcellular polar distribution of mitochondrial ribosomal RNA suggests the polarization of 4-cell blastomeres.MtrRNAs,including 12S rRNA and 16S rRNA,can only be detected by in situ hybridization in inner cells at morula and blastocyst stages.The distribution of Mtr rRNA did not coincide with mitochondria which was localized throughout the entire embryo.The amount of mitochondria and mitochondrial activity were not different in the sister blastomeres of 2-cell and 4-cell embryos.In our experiments,16S mtrRNA was shown to distribute extra-mitochondrially in the mouse preimplatation embryo using ISH immuno-electromicroscopy for 16S mtrRNA.We have determined the role of the extra-mitochondrial 16S mtrRNA in mouse embryogenesis and we found that 16S mtrRNA contributed to cell lineage determining.When we overexpressed 16S mtrRNA by injecting RNA into one blastomere of 2-cell embryo,the descendants of the injected cell mostly contributed to embryonic part of blastocyst.When we injected 16S mtrRNA antisense RNA into one blastomere of 2-cell embryo,the descendants of the injected cell distributed mixed in blastocyst.Meanwhile,we injected EGFP mRNA only into one blastomere of 2-cell embryo as control,the descendants of the injected cell tend to occupy either embryonic or abembryonic parts of the blastocyst and this distribution is consistent with the reported results of lineage tracing studies.Our studies show that the polarity of cells in mouse preimplantation embryo was not arise after compaction taking place at the 8-cell stage.The first difference between blastomeres has emerged as early as 2-cell stage,some genes were differently expressed between two sister blastomeres.In our experiment results,both 12S and 16S mitochondrial rRNAs were asymmetrically distributed in mouse embryos at all stages.MtrRNA level was different between two sister blastomeres and these difference can result in distinct cell lineages.When overexpressed 16S mtrRNA of one blastomere at 2-cell stage and the cell contributed all descendants to embryonic part of blastocyst embryo.When disrupted the function of 16S mtrRNA by injecting 16S antisense RNA into one blastomere of 2-cell embryo,cell lineage prepatternig at 2-cell stage was interrupted and descendants of injected cell distributed mixed in entire blastocyst embryo.