Project description:Upon 2-cell embryo splitting, individual blastomeres were compared and contrasted with each other respecting pair associations (e.g. blastomere '1a' and '1b' of embryo 1, '2a' and '2b' of embryo 2, and so forth) Transcriptome analysis followed by cluster analysis (Ward) was able to match a minority of the blastomeres with the correct sister blastomere
Project description:Following fertilization in mammals, it is generally accepted that totipotent cells are exclusive to the zygote and to each of the two blastomeres originating from the first mitotic division. We counter that this classic view needs to be revised, because we have presented compelling evidence the sister blastomeres are both totipotent in only a subset of 2-cell stage mouse embryos (PMID 28811525). Building on our previous findings, we here ask the question if the differences between sister blastomeres can be modulated experimentally. To this end, we separate the sister blastomeres, yielding monozygotic twins. We culture these twins in two different media (GM501 vs. Sage 1 step), to see if the mRNA ratios of twin blastocysts lie more far apart from each other when culture took place in different media as compared to culture in the same medium (GSE90674). Transcriptome analysis followed by intra-pair mRNA ratio analysis revealed differences between the blastocysts of the same monozygotic pair. Some of the differences were sensitive to the choice of culture conditions, while other differences were insensitive.
Project description:It remains an open question when and how the first cell fate decision is made in mammals. Using deep single-cell RNA-seq of matched sister blastomeres, we report highly reproducible interblastomere differences among ten 2-cell and five 4-cell mouse embryos. Inter-blastomere gene expression differences dominated between-embryo differences and noises, and were sufficient to cluster sister blastomeres into distinct groups. Dozens of protein-coding genes exhibited reproducible bimodal expression in sister blastomeres (0 vs. 1e3-1e6 of FPKM), which cannot be explained by random fluctuations. The protein expression of one of these bimodal genes, Gadd45a, exhibited clear inter-blastomeric contrasts. We traced some of the bimodal mRNA expressions to embryonic genome activation, and others to blastomere-specific RNA depletion. Inter-blastomere differences created co-expression gene networks that were much stronger and larger than those that can be possibly created by random noises. The highly correlated gene pairs at the 4-cell overlapped with those showing the same directions of differential expression between inner cell mass (ICM) and trophectoderm (TE). These data substantiate the hypothesis of inter-blastomere differences in 2- and 4-cell mouse embryos, and associate these differences with ICM/TE differences. 9 zygotes, 10 2-cell, and 5 4-cell mouse (C57BL/6) embryos were collected and multi-cell embryos were separated into blastomeres. 4 inner cell mass (ICM) and 3 trophectoderm (TE) samples are also extracted from mouse blastocysts. Transcriptome profiles for all samples are obtained via Smart-seq protocol.
Project description:The ability of sister blastomeres of 8-cell mouse embryos to produce ESC lines was measured, and the transcriptome of the Bm-mESCs lines generated was analyzed by RNA-seq.
Project description:It remains an open question when and how the first cell fate decision is made in mammals. Using deep single-cell RNA-seq of matched sister blastomeres, we report highly reproducible interblastomere differences among ten 2-cell and five 4-cell mouse embryos. Inter-blastomere gene expression differences dominated between-embryo differences and noises, and were sufficient to cluster sister blastomeres into distinct groups. Dozens of protein-coding genes exhibited reproducible bimodal expression in sister blastomeres (0 vs. 1e3-1e6 of FPKM), which cannot be explained by random fluctuations. The protein expression of one of these bimodal genes, Gadd45a, exhibited clear inter-blastomeric contrasts. We traced some of the bimodal mRNA expressions to embryonic genome activation, and others to blastomere-specific RNA depletion. Inter-blastomere differences created co-expression gene networks that were much stronger and larger than those that can be possibly created by random noises. The highly correlated gene pairs at the 4-cell overlapped with those showing the same directions of differential expression between inner cell mass (ICM) and trophectoderm (TE). These data substantiate the hypothesis of inter-blastomere differences in 2- and 4-cell mouse embryos, and associate these differences with ICM/TE differences.
Project description:Following fertilization in mammals, it is generally accepted that totipotent cells are exclusive to the zygote and to each of the two blastomeres originating from the first mitotic division. We counter that this classic view needs to be revised, because we have presented compelling evidence that the sister blastomeres are both totipotent in only a subset of 2-cell stage mouse embryos (PMID 28811525). Building on our previous findings, we here ask the question if the interblastomere differences depend - at least in part - on the contribution of sperm, since the area of sperm entry is inherited preferentially by one blastomere. To this end, we created sister 2-cell stage blastomeres without sperm entry point, by parthenogenesis. We compare the transcriptomes of the sister blastomeres, to see if the interblastomere mRNA differences observed after fertilization (GSE94050) are still there when the sperm entry point is missing.
Project description:We have examined whether twin blastomeres from 2-cell stage mouse embryos differ in mRNA content. Amplified mRNA from 12 blastomeres derived from six embryos approximately mid-way through their second cell cycle was analyzed. Probes displaying normalized values greater than 0.25 were selected and examined for consistent bias in expression within blastomere pairs. Although transcript content varied both between individual embryos and twin blastomeres, no consistent asymmetries were observed for the majority of genes. On the other hand, 769 genes displayed a greater than 1.4-fold difference in expression across 5 of 6 pairs of blastomeres and 178 genes differed across all 6 pairs. These genes separated into two groups by class discovery clustering. Of the 769 differentially expressed genes, 163 were significantly up- or down-regulated in one sister blastomere compared to the other. Transcripts encoding proteins implicated in RNA processing and cytoskeletal organization were highly represented among the most abundant, differentially distributed mRNA. We conclude that there are many differences that distinguish twin blastomeres derived from a single 2-cell stage embryo but that only a few of these differences are consistent across multiple pairs of embryos. We hypothesize that a stochastically-based lack of synchrony in cell cycle progression between the two cells might explain some or all of the asymmetries in transcript composition. 6 pairs of blastomeres were analyzed for a total of 12 samples.