Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Tracing the derivation of embryonic stem cells from the inner cell mass by single cell RNA-seq analysis

ABSTRACT: Here we modified a single cell whole transcriptome amplification method to make it capable of amplifying cDNAs as long as 3kb efficiently and unbiasedly. We combined this modified single cell cDNA amplification method with Applied Biosystems next generation sequencing SOLiD™ System to set up a single cell whole transcriptome assay. The modified amplification strategy allows us to amplify full-length cDNAs for most of the expressed genes. We show that it is feasible to get digital gene expression profiles at single cell resolution. This allows us to ask fundamental biological questions that could not be addressed previously, especially in the embyronic stem cell studues , and to understand transcriptome complexity at the resolution of a single cell. Four-cell stage embryos were recovered from MF1 females mated with MF1 male mice (Nagy et al. 2003). The zona pellucida was removed by treatment with acidic tyrode solution. The individual blastomeres were separated by gentle pipeting using a glass capillary. Mature oocytes were isolated from Dicer knockout [Dicer-/Flox, Zp3-Cre] and Ago2 knockout [Ago2-/Flox, Zp3-Cre] female mice (14,17). Cumulus cells were removed from oocytes by treatment with hyaluronidase. All the ‘mature oocytes’ mentioned in the text are ovulated mature oocytes. RNA-Seq from 13 single mouse ES cells, 9 single mouse E3.5 ICM, 3 Day3-Oct4+ outgrowth, 3 Day5-Oct4+ outgrowth, 3 Day5-Oct4- outgrowth and 3 mouse E.4.5 Epiblast

ORGANISM(S): Mus musculus

SUBMITTER: Catalin Barbacioru

PROVIDER: E-GEOD-20187 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Tracing the derivation of embryonic stem cells from the inner cell mass by single-cell RNA-Seq analysis.

Tang Fuchou F Barbacioru Catalin C Bao Siqin S Lee Caroline C Nordman Ellen E Wang Xiaohui X Lao Kaiqin K Surani M Azim MA

Cell stem cell 20100501 5

During the transition from the inner cell mass (ICM) cells of blastocysts to pluripotent embryonic stem cells (ESCs) in vitro, a normal developmental program is replaced in cells that acquire a capacity for infinite self-renewal and pluripotency. We explored the underlying mechanism of this switch by using RNA-Seq transcriptome analysis at the resolution of single cells. We detected significant molecular transitions and major changes in transcript variants, which include genes for general metabo ...[more]

PMID: 20452321

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Project description:Slimming is globally prevalent especially in young women, and it may contribute to the metabolic health of their offspring. Whereas some Lamarckian ideas about environmental inheritance have been dismissed, increasing evidence suggest that certain acquired traits can be transmitted to the next generation. It is therefore of great interest to determine how and to what extent a maternal lifestyle change contributes to their offspring. Here we show that enriched environment (EE) induced maternal slimming improves general health and reprograms metabolic gene expression in mice offspring. EE in mothers induced decreased body weight, adiposity, and improved glucose tolerance and insulin sensitivity. Relative to controls, their offspring exhibited improved general health such as reduced fat accumulation, enhanced metabolic parameters as well as glucose tolerance and insulin sensitivity. Maternal slimming altered the expression of 1,732 genes in the liver of offspring, with coherent downregulation of genes involved in lipid and cholesterol biosynthesis. Epigenomic profiling in offspring revealed numerous changes in cytosine methylation depending on maternal slimming, including hypermethylation of several genes involved in lipid biosynthesis, correlated with the downregulation of these genes. Maternal slimming also altered overall transcriptome patterns in mature oocytes, which contributes largely to the metabolic health and gene expression patterns in offspring. Overall, our studies suggest that maternal slimming have a beneficial role in regulating metabolic profiles in offspring, implying that it might be considered as a potential strategy to reverse the global prevalence of obesity and related metabolic syndromes. Examination of the effect of 2 different maternal lifestyles, control and slimming, on the mRNA expression in the mature oocytes of the female mice. Naturally ovulated mature oocytes (MII stage) were collected from 3 control and 3 slimming female F0 founders (3 oocytes per mouse, 9 oocytes for each group).

Project description:Mutation studies always defined the functions of the zona pellucida (ZP) as extracellular, namely: to encase the oocytes in ovarian follicles, to ensure species-specific sperm binding, and to dampen shear stress on the embryo surface. Therefore, mutations in the three ZP mouse genes ZP1, ZP2 or ZP3 cause primary infertility due to empty follicles, polyspermic fertilization or harmful contact between embryos and oviductal epithelium. However, the concepti of ZP2-null and ZP3-null oocytes were still unviable also when the defects were obviated by monospermic fertilization in vitro and blastocyst transfer to uterus (PMID 11245577). This suggests that the tasks of ZPs don’t end in the extracellular space as previously assumed, but there may be also intracellular functions yet to be discovered. The present study tested if experimentally induced degradation of intracellular ZP3 impacted on the development and transcriptome of mouse embryos. To this end we degraded ZP3 using its antibody in conjunction with the ubiquitin-protein ligase TRIM21. This method is known as 'Trim-away' (PMID 29153837). Briefly, in this method a cell (e.g. oocyte) expressing TRIM21 is supplied e.g. injected with a specific antibody to a protein of interest, in this case ZP3. As a result, the ternary complex (target protein-antibody-TRIM21) is destroyed in the proteasome. TRIM21 is here always to be understood as translation product of microinjected mCherry-Trim21 mRNA. We compared two experimental groups, as follows. Pronuclear-stage oocytes (B6C3F1 x CD1) were microinjected with approx. 100 picoliters of mix comprised of mCherry-Trim21 mRNA 0.2 mg/mL + buffer of ZP3 antibody + dextran beads 0.02 mg/mL as tracer, forming a group named 'Trim21 overexpression', in quadruplicate. As a reference, pronuclear-stage oocytes were microinjected with mCherry-Trim21 mRNA 0.2 mg/mL + anti-ZP3 antibody (Proteintech 21279-1-AP) 1 mg/mL + dextran beads, forming a group named 'Trim-away ZP3' group, in triplicate. To identify differently expressed genes we compared group 'Trim-away ZP3' with group 'Trim21 overexpression'. In addition, a single 'non-manipulated' sample was also incuded merely to confirm that Trim21 was detectable in the two experimental groups, but not in the non-microinjected embryos. On the day after microinjection, embryos were collected and lysed for transcriptome analysis. Those of group 'Trim21 overexpression' were at the 2-cell stage so as the non-manipulated embryos, whereas those of group 'Trim-away ZP3' were arrested at the 1-cell stage. Transcriptome analysis revealed that embryos of group 'Trim-away ZP3' and 'group 'Trim21 overexpression' differed in the expression of 197 of 11137 genes (t test, FDR<0.05). The data support a conclusion that ZP3 found inside the embryo was not merely a remnant from oogenesis, but served an intracellular, post-fertilization role during mouse preimplantation development.

Project description:Mutation studies always defined the functions of the zona pellucida (ZP) as extracellular, namely: to encase the oocytes in ovarian follicles, to ensure species-specific sperm binding, and to dampen shear stress on the embryo surface. Therefore, mutations in the three ZP mouse genes ZP1, ZP2 or ZP3 cause primary infertility due to empty follicles, polyspermic fertilization or harmful contact between embryos and oviductal epithelium. However, the concepti of ZP2-null and ZP3-null oocytes were still unviable also when the defects were obviated by monospermic fertilization in vitro and blastocyst transfer to uterus (PMID 11245577). This suggests that the tasks of ZPs don’t end in the extracellular space as previously assumed, but there may be also intracellular functions yet to be discovered. The present study tested if experimentally induced degradation of intracellular ZP3 impacted on the development and transcriptome of mouse embryos. To this end we degraded ZP3 using its antibody in conjunction with the ubiquitin-protein ligase TRIM21. This method is known as 'Trim-away' (PMID 29153837). Briefly, in this method a cell (e.g. oocyte) expressing TRIM21 is supplied e.g. injected with a specific antibody to a protein of interest, in this case ZP3. As a result, the ternary complex (target protein-antibody-TRIM21) is destroyed in the proteasome. TRIM21 is here always to be understood as translation product of microinjected mCherry-Trim21 mRNA. We compared two experimental groups, as follows. Pronuclear-stage oocytes (B6C3F1 x CD1) were microinjected with approx. 100 picoliters of mix comprised of mCherry-Trim21 mRNA 0.2 mg/mL + anti-ZP3 antibody (Proteintech 21279-1-AP) 1 mg/mL + dextran beads 0.02 mg/mL, forming a group named 'Trim-away ZP3' group, in triplicate. As a reference, pronuclear-stage oocytes were microinjected with the same mixture as above, except that the antibody buffer was used in lieu of the antibody itself, in triplicate, forming a group named ‚no Trim'. To identify differently expressed genes we compared group 'Trim-away ZP3' with group ‘no Trim’. Ten hours after microinjection, embryos were collected and lysed for transcriptome analysis. Transcriptome analysis revealed that embryos of group 'Trim-away ZP3' and group ‘no Trim' differed in gene expression and were resolved in principal component analysis. The data support a conclusion that ZP3 found inside the embryo was not merely a remnant from oogenesis, but served an intracellular, post-fertilization role during mouse preimplantation development.

Dataset Information

Tracing the derivation of embryonic stem cells from the inner cell mass by single cell RNA-seq analysis

Publications

Tracing the derivation of embryonic stem cells from the inner cell mass by single-cell RNA-Seq analysis.

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