Project description:Expression of germ cell nuclear factor (GCNF, Nr6a1), an orphan member of the nuclear receptor gene family of transcription factors, during gastrulation and neurulation is critical for normal embryogenesis in mice. Gcnf represses the expression of the POU domain transcription factor Oct4 (Pou5f1) during mouse post-implantation development. Although Gcnf expression is not critical for the embryonic segregation of the germ cell lineage, we found that sexually dimorphic expression of Gcnf in germ cells correlates with the expression of pluripotency-associated genes, such as Oct4, Sox2, and Nanog, as well as the early meiotic marker gene Stra8. To elucidate the role of Gcnf during mouse germ cell differentiation, we generated an ex vivo Gcnf-knockdown model in combination with a regulated CreLox mutation of Gcnf. Lack of Gcnf impairs normal spermatogenesis and oogenesis in vivo, as well as the derivation of germ cells from embryonic stem cells (ESCs) in vitro. Inactivation of the Gcnf gene in vivo leads to loss of repression of Oct4 expression in both male and female gonads. For RNA isolation, PGCs were sorted by FACS, collected by centrifugation, lysed in RLT buffer (QIAGEN), and processed using the RNeasy micro and mini kits with on-column DNase digestion as per the manufacturer’s instructions. Integrity of RNA samples was quality checked using a 2100 Bioanalyzer (Agilent). When possible, 300 ng of total RNA per sample was used as starting material for linear amplification (Illumina TotalPrep RNA Amplification Kit, Ambion), which involved synthesis of T7-linked double-stranded cDNA and 14 hr of in-vitro transcription incorporating biotin-labeled nucleotides. Purified and labeled cRNA was then quality checked on a 2100 Bioanalyser and hybridized as biological or technical duplicates for 18 hr onto MouseRef-8 v2 gene expression BeadChips (Illumina), following the manufacturer's instructions. After being washed, the chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using an iScan reader and accompanying software. Bead intensities were mapped to gene information using BeadStudio 3.2 (Illumina). Background correction was performed using the Affymetrix Robust Multi-array Analysis (RMA) background correction model. Variance stabilization was performed using log2 scaling, and gene expression normalization was calculated with the quantile method implemented in the lumi package of R-Bioconductor. Data post-processing and graphics were performed with in-house developed functions in Matlab. 8 samples were analyzed: ESC++, ESC control Oct4-GFP, 1 replicate ESC--, ESC Gcnf -/- Oct4-GFP, 1 replicate D15++, Day 15 in vitro derived PGCs from GCNF +/+ Oct4-GFP ESC, 1 replicate D15--, Day 15 in vitro derived PGC from GCNF -/- Oct4-GFP ESC, 1 replicate D12++, Day 12 in vitro derived PGC from GCNF +/+ Oct4-GFP ESC, 1 replicate D12--, Day 12 in vitro derived PGC from GCNF -/- Oct4-GFP ESC, 1 replicate EpiSC, EpiESC, 2 replicates

Project description:Several Sox family members, small molecules, and inhibitors of the TGF-β pathway can replace exogenous Sox2 during the generation of induced pluripotent stem cells (iPSCs), suggesting that SOX2 is dispensable for initiation of reprogramming. However, the time point at which the endogenous Sox2 locus is activated in these conditions was not thoroughly evaluated. To identify the Sox2-independent reprogramming steps, we excluded Sox2 from the reprogramming cocktail and used Sox2-deficient mouse embryonic fibroblasts. We show that reprogramming is initiated in the absence of SOX2 as shown by formation of pre-iPSC colonies that have undergone a mesenchymal-to-epithelial transition and that have activated early reprogramming markers. However, these iPSC-like intermediates cannot progress through the final stabilization phase of reprogramming in the absence of SOX2. Overall, our results provide a better understanding of the molecular events underlying the induction of pluripotency and of the role that SOX2 plays in the process. Both pLV-tetO-E1A-2A-Cherry and pLV-tetO-Cherry lentiviral constructs were cloned in house and defective-lentiviral particles were produced as previously described (Han et al. 2013). Next, 5x10 4 ESCs containing a tetracycline-inducible transactivator (irtTA-VP16-GBD, Anastassiadis et al. 2002) plus an Oct4-GFP reporter transgene (Yoshimizu et al. 1999) were transduced twice using 20 µl of replication-defective lentiviral particles plus polybrene (4 µg/ml, Sigma). Cells were washed 24 h after transduction and subsequently induced with doxycycline (1 µg/ml, Sigma) and dexamethasone (10-7 M, Sigma). Samples were harvested at several time-points, 0, 3, 6, 9, 12, 24, and 48 hours after induction, and used for microarray analysis as previously reported (Tapia et al. 2012). Cells transduced with pLV-tetO-Cherry and induced for 48 h were used as control. RNA wasprocessed using the RNeasy micro and mini kits with on-column DNase digestion of QIAGEN as per the manufacturer’s instructions. Integrity of RNA samples was quality checked using a 2100 Bioanalyzer (Agilent). When possible, 300 ng of total RNA per sample was used as starting material for linear amplification (Illumina TotalPrep RNA Amplification Kit, Ambion), which involved synthesis of T7-linked double-stranded cDNA and 14 hr of in-vitro transcription incorporating biotin-labeled nucleotides. Purified and labeled cRNA was then quality checked on a 2100 Bioanalyser and hybridized as biological or technical duplicates for 18 hr onto MouseRef-8 v2 gene expression BeadChips (Illumina), following the manufacturer's instructions. After being washed, the chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using an iScan reader and accompanying software. Bead intensities were mapped to gene information using BeadStudio 3.2 (Illumina). Background correction was performed using the Affymetrix Robust Multi-array Analysis (RMA) background correction model. Variance stabilization was performed using log2 scaling, and gene expression normalization was calculated with the quantile method implemented in the lumi package of R-Bioconductor. Data post-processing and graphics were performed with in-house developed functions in Matlab.

Project description:Human and mouse somatic cells can be reprogrammed by the combination of Oct4, Sox2, Klf4, and c-Myc, but the efficiency of reprogramming is low. To better understand the process of reprogramming we sought to identify factors that mediate reprogramming at higher efficiency. For this we established an assay to screen nuclear fractions from the extracts of pluripotent cells based on Oct4 reactivation. We identified components of the ATP-dependent SWI/SNF chromatin-remodeling complex, which when used along with the above four factors increase reprogramming efficiency by five-fold and improve the quality of the reprogrammed cells. These cells were found to be capable of germline transmission and exhibited pluripotency according to gene expression and in vivo and in vitro assays. SWI/SNF was found to replace c-Myc and mediate its effects by facilitating recruitment of Oct4 on target promoters during reprogramming. Thus, somatic cell reprogramming using chromatin-remodeling molecules represents an efficient method of generating reprogrammed cells. DNA-free RNA samples to be hybridized on Illumina expression BeadChips were processed using a linear amplification kit (Ambion) (generating IVT duration: 12h). cRNA samples were quality-checked on a 2100 Bioanalyzer (Agilent) and hybridized as biological triplicates onto MouseWG-6 V2 chips as recommended and using materials / reagents provided by the manufacturer (hybridization time: 18h). The Myc probe on the V2 arrays was found to be defective. The bead intensities were mapped to gene information using BeadStudio 3.2 (Illumina), background correction was performed using the Affymetrix Robust Multi-array Analysis (RMA) background correction model, variance stabilization was performed using log2 scaling, and gene expression normalization was calculated with the quantile method implemented in the lumi package of R-Bioconductor. Data post-processing and graphics were performed with in-house developed functions in Matlab. Hierarchical clusters of genes and samples were performed with a one minus correlation metric and the average (unweighted pair group) linkage method.

Project description:The molecular mechanisms underlying the commitment of cells to the germ cell lineage and the subsequent formation of germ cells during mammalian development remain poorly understood due to an inability to obtain sufficient amounts of cellular materials to conduct thorough in-vitro analyses. Although primordial germ cells (PGCs) have been generated and isolated from embryonic stem cells (ESCs) in vitro, concurrent expression of several cell surface receptors and transcription factors, at various levels, confounds the identification of these cells. To date, only a few genes that mark the onset of germ cell commitment to the epiblast, including tissue nonspecific alkaline phosphatase (TNAP), Blimp1, Stella, and Fragilis, have been used with some degree of success to detect PGC formation in in-vitro model systems. In light of the current literature, the generation of an unlimited supply of germ cells and gametes from pluripotent stem cells would greatly facilitate studies into reproductive development. To accomplish this, we would need to discover novel markers that are specifically expressed in germ cells. Here we identified four genes that are specifically expressed in male and female germ cells, both in vivo and in vitro, but are not expressed in somatic tissues or ESCs. Expression of these genes therefore allows us to distinguish committed germ cells from undifferentiated pluripotent cell populations, a prerequisite for the successful derivation of germ cells and gametes in vitro. For RNA isolation, PGCs were sorted by FACS, collected by centrifugation, lysed in RLT buffer (QIAGEN), and processed using RNeasy Micro kits with on-column DNase digestion as per the manufacturer's instructions. Integrity of RNA samples was quality-checked using a 2100 Bioanalyzer (Agilent). If possible, 300 ng of total RNA per sample was used as starting material for linear amplification (Illumina TotalPrep RNA amplification kit, Ambion), which involved synthesis of T7-linked double-stranded cDNA and 14 hrs of in-vitro transcription incorporating biotin-labeled nucleotides. Purified and labeled cRNA was then quality-checked on a 2100 Bioanalyser, and hybridized as biological or technical duplicates for 18 hrs onto MouseRef-8 v2 gene expression BeadChips (Illumina), following the manufacturer's instructions. After being washed, the chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using the iScan reader and accompanying software. 44 samples were analyzed. ESCm: OG2 male Embryonic Stem Cell, +/+ Oct4-GFP ES cells, 1 biological rep 11.5: 11.5 embryonic day PGC (Primordial Germ Cell), 2 biological reps 12.5F: 12.5 embryonic day female PGC (Primordial Germ Cell), 2 biological reps 13.5F: 13.5 embryonic day female PGC (Primordial Germ Cell), 2 biological reps 14.5F: 14.5 embryonic day female PGC (Primordial Germ Cell), 2 biological reps 15.5F: 15.5 embryonic day female PGC (Primordial Germ Cell), 2 biological reps 16.5F: 16.5 embryonic day female PGC (Primordial Germ Cell), 2 biological reps 17.5F: 17.5 embryonic day female PGC (Primordial Germ Cell), 2 biological reps 18.5F: 18.5 embryonic day female PGC (Primordial Germ Cell), 2 biological reps 12.5M: 12.5 embryonic day male PGC (Primordial Germ Cell), 2 biological reps 13.5M: 13.5 embryonic day male PGC (Primordial Germ Cell), 2 biological reps 14.5M: 14.5 embryonic day male PGC (Primordial Germ Cell), 2 biological reps 15.5M: 15.5 embryonic day male PGC (Primordial Germ Cell), 2 biological reps 16.5M: 16.5 embryonic day male PGC (Primordial Germ Cell), 2 biological reps 17.5M: 17.5 embryonic day male PGC (Primordial Germ Cell), 2 biological reps 18.5M: 18.5 embryonic day male PGC (Primordial Germ Cell), 2 biological reps 8M: 8 day after birth male PGC (Primordial Germ Cell), 2 biological reps 10M: 10 day after birth male PGC (Primordial Germ Cell), 2 biological reps 12M: 12 day after birth male PGC (Primordial Germ Cell), 2 biological reps 14M: 14 day after birth male PGC (Primordial Germ Cell), 1 biological rep 16M: 16 day after birth male PGC (Primordial Germ Cell), 2 biological reps 18M: 18 day after birth male PGC (Primordial Germ Cell), 2 biological reps 20M: 20 day after birth male PGC (Primordial Germ Cell), 2 biological reps

Project description:Human and mouse somatic cells can be reprogrammed by the combination of Oct4, Sox2, Klf4, and c-Myc, but the efficiency of reprogramming is low. To better understand the process of reprogramming we sought to identify factors that mediate reprogramming at higher efficiency. For this we established an assay to screen nuclear fractions from the extracts of pluripotent cells based on Oct4 reactivation. We identified components of the ATP-dependent SWI/SNF chromatin-remodeling complex, which when used along with the above four factors increase reprogramming efficiency by five-fold and improve the quality of the reprogrammed cells. These cells were found to be capable of germline transmission and exhibited pluripotency according to gene expression and in vivo and in vitro assays. SWI/SNF was found to replace c-Myc and mediate its effects by facilitating recruitment of Oct4 on target promoters during reprogramming. Thus, somatic cell reprogramming using chromatin-remodeling molecules represents an efficient method of generating reprogrammed cells. DNA-free RNA samples to be hybridized on Illumina expression BeadChips were processed using a linear amplification kit (Ambion) (generating IVT duration: 12h). cRNA samples were quality-checked on a 2100 Bioanalyzer (Agilent) and hybridized as biological triplicates onto MouseWG-6 V2 chips as recommended and using materials / reagents provided by the manufacturer (hybridization time: 18h). The Myc probe on the V2 arrays was found to be defective. The bead intensities were mapped to gene information using BeadStudio 3.2 (Illumina), background correction was performed using the Affymetrix Robust Multi-array Analysis (RMA) background correction model, variance stabilization was performed using log2 scaling, and gene expression normalization was calculated with the quantile method implemented in the lumi package of R-Bioconductor. Data post-processing and graphics were performed with in-house developed functions in Matlab. Hierarchical clusters of genes and samples were performed with a one minus correlation metric and the average (unweighted pair group) linkage method. 4 samples were analyzed, each one them by triplicate. ESC: Mouse Embryonic Stem Cells (OG2 mix female and male) MEF: Mouse Embryonic Fibroblasts (OG2 male) iPS4F: Mouse induced Pluripotent Stem cells with 4 factors (Oct4, Sox2, Klf4, c-myc) iPS6F: Mouse induced Pluripotent Stem cells with 6 factors (Oct4, Sox2, Klf4, c-myc, Brg1, Baf155)

Project description:Oct4 is considered a master transcription factor for pluripotent cell self-renewal and embryo development. It primarily collaborates with other transcriptional factors or coregulators to maintain pluripotency. However, it is still unclear how Oct4 interacts with its partners. Here, we show that the Oct4 linker interface mediates competing and balanced Oct4 protein interactions which are crucial for maintaining pluripotency. Linker mutant ESCs maintain the key pluripotency genes expression, but show decreased expression of self-renewal genes and increased expression of differentiation genes which result in impaired ESCs self-renewal and early embryonic lethality. Linker mutation dose not affect Oct4 genomic binding and transactivation potential, but breaks the balanced Oct4 interactome. In mutant ESCs, the interaction between Oct4 and Klf5 was decreased, but Oct4-Cbx1, Ctr9, Cdc73 interactions were increased which disrupt the epigenetic state of ESCs. Overexpression of Klf5 or knockdown Cbx1, Cdc73 rescue the cellular phenotype of linker mutant ESCs by rebalancing Oct4 interactome indicating that different partners interact with Oct4 competitively. Thus, by showing how Oct4 interacts with different partners, we provide novel molecular insights to explain how Oct4 contributes to the maintenance of pluripotency. RNA samples to be analyzed by qRT-PCR and microarrays were prepared using RNeasy Mini Kit (QIAGEN) with on-column DNA digestion. Complementary DNA for qRT-PCR was synthesized with the M-MLV Reverse Transcriptase (Affymetrix). Transcript levels were determined using ABI PRISM Sequence Detection System 7900 and gene expression was normalized to the housekeeping gene Gapdh. 300 ng of total RNA per sample was used as input using a linear amplification protocol (Ambion) which involved synthesis of T7-linked double-stranded cDNA and 12 hours of in vitro transcription incorporating biotin-labeled nucleotides. Purified and labeled cRNA was then hybridized for 18 h onto MouseRef-8 v2 expression BeadChips (Illumina) following the manufacturer’s instructions. After washing, chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using the iScan reader (Illumina). The bead intensities were mapped to the corresponding gene information using BeadStudio 3.2 (Illumina) and background correction was performed using the Affymetrix Robust Multi-array Analysis (RMA) background correction model (Irizarry et al., 2003). Variance stabilization was performed using the log2 scaling, and gene expression normalization was calculated with the quantile method implemented in the lumi package of R-Bioconductor. Data post-processing and graphics were performed with in-house developed functions in MATLAB. Hierarchical clusters of genes and samples were performed with the one minus the sample correlation metric and the Unweighted Pair-Group Method using Average (UPGMA) linkage method.

Project description:To study the mechanisms of pluripotency induction, we compared gene expression in pluripotent embryonic germ cells (EGCs) and unipotent primordial germ cells (PGCs). We found 11 genes ≥1.5-fold overexpressed in EGCs. None of the genes identified were Yamanaka's genes, but instead related to glycolytic metabolism. The prospect of pluripotency induction by cell metabolism manipulation was investigated by hypoxic culturing. We demonstrate that hypoxia alone induces reprogramming in PGCs, giving rise to hypoxia-induced EGC-like cells (hiEGL), which differentiate into cells of the three germ layers in vitro, and contribute to the ICM in vivo, demonstrating pluripotency. The mechanism of hypoxia induction involves HIF1? stabilization and Oct4 deregulation. However, hiEGL cannot be passaged long term. Self-renewal capacity is not achieved by hypoxia likely due to lack of upregulation of c-Myc and Klf4. Gene expression analysis of hypoxia signalling suggests that hiEGLs have not reached the stabilization phase of cell reprogramming. The data suggests that the properties of stemness, pluripotency and selfrenewal, are differentially regulated in primordial germ cell reprogramming induced by hypoxia. 8.5 dpc Oct4-GFP PGCs cultured in hypoxia conditions for 6 days were isolated by FACS. To eliminate the possibility of differences due solely to anaerobic respiration, TGC-10 EGC line was exposed to the hypoxia-mimetic agent Cl2Co at 150 microM for four hours prior to RNA isolation. RNA samples to be analyzed by microarrays were prepared using Qiagen RNeasy columns with on-column DNA digestion. 300 ng of total RNA per sample was used as input into a linear amplification protocol (Tetu-Bio), which involved synthesis of T7-linked double-stranded cDNA and 12 hours of in vitro transcription incorporating biotin-labelled nucleotides. Purified and labeled cRNA was then hybridized for 18h onto MouseRef-8 v2 expression BeadChips (Illumina) following the manufacturer's instructions. After washing, chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using the iScan reader (Illumina) and accompanying software. Samples were exclusively hybridized as biological replicates. The bead intensities were mapped to gene information using BeadStudio 3.2 (Illumina). Background correction was performed using the Affymetrix Robust Multi-array Analysis (RMA) background correction model. Variance stabilization was performed using the log2 scaling and gene expression normalization was calculated with the method implemented in the lumi package of RBioconductor. Data post-processing and graphics were performed with in-house developed functions in Matlab. Hierarchical clustering of genes and samples was performed with one minus correlation metric and the unweighted average distance (UPGMA) (also known as group average) linkage method.

Project description:To study the mechanisms of pluripotency induction, we compared gene expression in pluripotent embryonic germ cells (EGCs) and unipotent primordial germ cells (PGCs). We found 11 genes ≥1.5-fold overexpressed in EGCs. None of the genes identified were Yamanaka's genes, but instead related to glycolytic metabolism. The prospect of pluripotency induction by cell metabolism manipulation was investigated by hypoxic culturing. We demonstrate that hypoxia alone induces reprogramming in PGCs, giving rise to hypoxia-induced EGC-like cells (hiEGL), which differentiate into cells of the three germ layers in vitro, and contribute to the ICM in vivo, demonstrating pluripotency. The mechanism of hypoxia induction involves HIF1? stabilization and Oct4 deregulation. However, hiEGL cannot be passaged long term. Self-renewal capacity is not achieved by hypoxia likely due to lack of upregulation of c-Myc and Klf4. Gene expression analysis of hypoxia signalling suggests that hiEGLs have not reached the stabilization phase of cell reprogramming. The data suggests that the properties of stemness, pluripotency and selfrenewal, are differentially regulated in primordial germ cell reprogramming induced by hypoxia. 8.5 dpc Oct4-GFP PGCs cultured in hypoxia conditions for 6 days were isolated by FACS. To eliminate the possibility of differences due solely to anaerobic respiration, TGC-10 EGC line was exposed to the hypoxia-mimetic agent Cl2Co at 150 microM for four hours prior to RNA isolation. RNA samples to be analyzed by microarrays were prepared using Qiagen RNeasy columns with on-column DNA digestion. 300 ng of total RNA per sample was used as input into a linear amplification protocol (Tetu-Bio), which involved synthesis of T7-linked double-stranded cDNA and 12 hours of in vitro transcription incorporating biotin-labelled nucleotides. Purified and labeled cRNA was then hybridized for 18h onto MouseRef-8 v2 expression BeadChips (Illumina) following the manufacturer's instructions. After washing, chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using the iScan reader (Illumina) and accompanying software. Samples were exclusively hybridized as biological replicates. The bead intensities were mapped to gene information using BeadStudio 3.2 (Illumina). Background correction was performed using the Affymetrix Robust Multi-array Analysis (RMA) background correction model. Variance stabilization was performed using the log2 scaling and gene expression normalization was calculated with the method implemented in the lumi package of RBioconductor. Data post-processing and graphics were performed with in-house developed functions in Matlab. Hierarchical clustering of genes and samples was performed with one minus correlation metric and the unweighted average distance (UPGMA) (also known as group average) linkage method. 14 samples were analyzed EG: pluripotent Embryonic Germ Cells (EGCs), 2 replicates hiEGL1: hypoxia-induced EGC-like cells (hiEGL), 2 replicates hiEGL2: hypoxia-induced EGC-like cells (hiEGL), 2 replicates ESCm: Embryonic Stem Cells (ESCs) male, 2 replicates ESCf: Embryonic Stem Cells (ESCs) female, 2 replicates EpiT9: Epiblast Stem Cells T9, 1 replicate EpiE3: Epiblast Stem Cells E3, 1 replicate PGC11.5: unipotent Primordial Germ Cells (PGCs) from dpc 11.5 , 2 replicates

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Germ cell nuclear factor regulates gametogenesis in developing gonads

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