Quantitative Dynamics of Chromatin-state Reprogramming for the Mouse Germ-Cell Specification Pathway In Vitro
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE60018 | GEO | 2015/03/20
SECONDARY ACCESSION(S): PRJNA257283
REPOSITORIES: GEO
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Project description:The germ cell lineage ensures the continuity of life through the generation of male and female gametes, which unite to form a totipotent zygote. We have established a culture system that recapitulates the mouse germ-cell specification pathway: Using cytokines, embryonic stem cells (ESCs)/induced pluripotent stem cells (iPSCs) are induced into epiblast-like cells (EpiLCs) and then into primordial germ cell-like cells (PGCLCs) with capacity both for spermatogenesis and oogenesis, creating an opportunity for understanding and regulating mammalian germ cell development in both sexes in vitro. Here we show that, without cytokines, simultaneous over-expression of three transcription factors (TFs), Blimp1 (also known as Prdm1), Prdm14 and Tfap2c (also known as AP2γ), directs EpiLCs, but not ESCs, swiftly and highly efficiently into a PGC state with endogenous transcription circuitry. The induction of the PGC state on EpiLCs minimally requires Prdm14 but not Blimp1 or Tfap2c. The TF-induced PGC state reconstitutes key transcriptome and epigenetic reprogramming in PGCs, but bypasses a mesodermal program that accompanies PGC specification in vivo and in vitro by cytokines including BMP4. Importantly, the TF-induced PGC-like cells robustly contribute to spermatogenesis and fertile offspring. Our findings provide not only a novel insight into the transcriptional logic that creates a germ cell state, but also a foundation for the TF-based reconstitution and regulation of mammalian gametogenesis. Aim of this analysis is characterization of transcription factor-induced primordial germ cells (TF-PGCLCs) compared with cytokine-induced primordial germ cells (Ck-PGCLCs) (Hayashi et al., 2011, Cell), epiblast-like cells (EpiLCs) (Hayashi et al., 2011, Cell), and embryonic stem cells (ESCs) and identification of genes differentially expressed among them. TF-PGCLCs induced by multiple combinations of TFs (Blimp1 (B), Prdm14 (P14), and Tfap2c (A) (BP14A), BP14, P14A, P14) on day 2 and 4 (for BP14A cells) of the induction were also compared. Parental clone without exogenous TFs cultured with doxycycline, are also included as a negative control. Ck-PGCLCs day 2 and day 4 samples, which are previously unreported, EpiLCs and ESCs used in this study were also included. Overexpression of exogenous three TFs in ESCs yields stella-ECFP (SC) positive cells, which were sorted and included in the analysis. cDNA samples, prepared from approximately 20,000 cells, were amplified with a quantitative global PCR method (Kurimoto et al., 2006, Nucleic Acids Research). Two biological duplicates for each cell type were analyzed. Samples from GSE30056 were also included and reanalysed (GSM1070855-GSM1070864).
2013-08-04 | E-GEOD-43775 | biostudies-arrayexpress
Project description:The generation of properly functioning gametes in vitro, a key goal in developmental/reproductive biology, requires multi-step reconstitutions of complex germ cell development. Based on the logic of primordial germ cell (PGC)-specification, we demonstrate here the generation of PGC-like cells (PGCLCs) in mice with robust capacity for spermatogenesis from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) through epiblast-like cells (EpiLCs), a cellular state highly similar to pre-gastrulating epiblasts, but distinct from epiblast stem cells (EpiSCs). The global transcription profiles, epigenetic reprogramming, and cellular dynamics during PGCLC induction from EpiLCs are a meticulous capture of those associated with PGC specification from the epiblasts. Furthermore, we identify Integrin-beta 3 and SSEA1 as markers that purify PGCLCs with spermatogenic capacity free from tumorigenic undifferentiated cells. With the reconstitution of PGC specification pathway from the naive inner cell mass state, our study defines a paradigm for the essential step of in vitro gametogenesis. We performed this analysis to reveal the characters of the cells that we created in this study, epiblast-like cells (EpiLCs) and primordial germ cells-like cells (PGCLCs). Because EpiLCs were induced from embryonic stem cells (ESCs), and equivalent to pre-gastrulating epiblast (embryonic day [E] 5.5-6.0) in vivo (embryo), ESCs and epiblast were included in this analysis. Epiblast stem cells (EpiSCs) are a culture cell type derived from epiblast, and were also included. PGCLCs were supposed to be equivalent to E9.5 PGCs based on reporter fluorescent transgene expressions and epigenetic properties, and therefore E9.5 PGCs were also inckuded in this analysis. Because epiblast and E9.5 PGCs are of a small number of cells in embryos (a few hundred to thousand cells), cDNAs were amplified with a quantitative global PCR method (Kurimoto et al., 2006, Nucleic Acids Research) for microarray analyses. We took two biological replicate for each cell type.
2011-08-04 | E-GEOD-30056 | biostudies-arrayexpress
Project description:The germ cell lineage ensures the continuity of life through the generation of male and female gametes, which unite to form a totipotent zygote. We have established a culture system that recapitulates the mouse germ-cell specification pathway: Using cytokines, embryonic stem cells (ESCs)/induced pluripotent stem cells (iPSCs) are induced into epiblast-like cells (EpiLCs) and then into primordial germ cell-like cells (PGCLCs) with capacity both for spermatogenesis and oogenesis, creating an opportunity for understanding and regulating mammalian germ cell development in both sexes in vitro. Here we show that, without cytokines, simultaneous over-expression of three transcription factors (TFs), Blimp1 (also known as Prdm1), Prdm14 and Tfap2c (also known as AP2?), directs EpiLCs, but not ESCs, swiftly and highly efficiently into a PGC state with endogenous transcription circuitry. The induction of the PGC state on EpiLCs minimally requires Prdm14 but not Blimp1 or Tfap2c. The TF-induced PGC state reconstitutes key transcriptome and epigenetic reprogramming in PGCs, but bypasses a mesodermal program that accompanies PGC specification in vivo and in vitro by cytokines including BMP4. Importantly, the TF-induced PGC-like cells robustly contribute to spermatogenesis and fertile offspring. Our findings provide not only a novel insight into the transcriptional logic that creates a germ cell state, but also a foundation for the TF-based reconstitution and regulation of mammalian gametogenesis. Aim of this analysis is identification of genes whose expression was altered by each of key transcription factor for transcription factor-induced primordial germ cells (TF-PGCLCs) induction (Blimp1 (B), Prdm14 (P14), and Tfap2c (A)). Both of epiblast-like cells (EpiLCs) (Hayashi et al., 2011, Cell) and aggregates of EpiLCs cultured with doxycycline on day 1 were harvested for 5 cell lines, including BP14A (Clone #3-3), B (#2-4), P14 (#7-109), A (#8-2), and the parental clone (BVSCR26rtTA embryonic stem cells). Total RNA was isolated and analyzed. Two biological duplicates for each cell type were included.
2013-08-04 | E-GEOD-46854 | biostudies-arrayexpress
Project description:Nanog, a core pluripotency factor in the inner cell mass of blastocysts, is also expressed in unipotent primordial germ cells (PGC) in mice1, where its precise role is yet unclear2-4. We investigated this in an in vitro model, where naïve pluripotent embryonic stem cells (ESCs) cultured in bFGF/ActivinA develop as epiblast-like cells (EpiLCs), and gain competence for PGC-like fate5. Consequently, bone morphogenetic protein (BMP4), or ectopic expression of key germline transcription factors Prdm1/ Prdm14/ Tfap2c, directly induce PGC-like cells (PGCLCs) in EpiLCs, but not in ESCs6-8. Here we report an unexpected discovery that Nanog alone can induce PGCLCs in EpiLCs, independently of BMP4. We propose that following the dissolution of the naïve ESC pluripotency network during establishment of EpiLCs9,10, the epigenome is reset for cell fate determination. Indeed, we found genome-wide changes in NANOG binding pattern between ESCs and EpiLCs, indicating epigenetic resetting of regulatory elements. Accordingly, we show that NANOG can bind and activate enhancers of Prdm1 and Prdm14 in EpiLCs in vitro; BLIMP1 (encoded by Prdm1) then directly induces Tfap2c. Furthermore, while SOX2 and NANOG promote the pluripotent state in ESCs, they show contrasting roles in EpiLCs since Sox2 specifically represses PGCLC induction by Nanog. This study demonstrates a broadly applicable mechanistic principle for how cells acquire competence for cell fate determination, resulting in the context-dependent roles of key transcription factors during development. To characterize Nanog-induced Primordial Germ cell-like cells (PGCLCs), we performed expression analysis of Nanog-induced Day4 PGCLCs compared to male mouse Embryonic Stem Cells (mESCs) and male Day4 PGCLCs which were induced by cytokines. mESCs were maintained in N2B27 2i(CHIR99021 3 µM, PD0325901 1 µM) LIF(1000 U/ml) medium and Day4 PGCLCs were induced in GK15 medium with Nanog induction (0.7 µg/ml) or cytokines (BMP4 500 ng/ml, BMP8A 500 ng/ml, SCF 100 ng/ml, EGF 50 ng/ml and LIF 1000U/ml) as previously reported (Hayashi K et al., Cell, 2011).
2016-01-11 | E-GEOD-61924 | biostudies-arrayexpress
Project description:The germ cell lineage ensures reproduction and heredity in metazoans. Primordial germ cells (PGCs) in mice are induced in pluripotent epiblast cells by BMP4 and WNT3, yet their mechanism of action remains elusive. Here, using in vitro PGC specification system, we show that WNT3, but not BMP4, induces many transcription factors associated with mesoderm in epiblast-like cells (EpiLCs) through beta-CATENIN. Among these, T (BRACHYURY), a classical and conserved mesodermal factor, was essential for robust activation of Blimp1 and Prdm14, two of the germline determinants. T, but not SMAD1 or beta-CATENIN/TCF1, binds distinct regulatory elements of both Blimp1 and Prdm14, and directly up-regulates these genes without BMP4 and WNT3. Without BMP4, a program induced by WNT3 prevents T from activating Blimp1 and Prdm14, demonstrating that BMP4 is permissive for PGC specification. These findings establish a fundamental role of a mesodermal gene in PGC specification, a potentially evolutionarily conserved mechanism across metazoans. Wnt3 (+/+) and Wnt3 (-/-) mouse embryonic stem cells (mESCs) bearing the BV transgenes expressing membrane-targeted Venus under the control of Blimp1 regulatory elements (Blimp1-mVenus: BV; PMID 18583473) were established and maintained. Epiblast-like cells (EpiLCs) were then induced from the established ES lines, followed by the cytokine stimulation (No cytokines [negative control], BMP4, Wnt3a, BMP4+Wnt3a) for different periods (0h, 12h, 24h, 36h).
2013-12-11 | E-GEOD-49689 | biostudies-arrayexpress
Project description:Transcriptomes of mouse E12.5 primordial germ cells (PGCs), primordial germ cell-like cells (PGCLCs) isolated from 6-day culture embryoid bodies, and the precursor pluripotent stem cells [embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs)] and epiblast-like cells (EpiLCs) Total RNA was isolated from FACS-enriched, SSEA1+/CD61+ double-positive PGCs and PGCLCs. RNA was also isolated from ESC, iPSC, and EpiLC cultured without enrichment. Transcriptomes were determined using Affymetrix microarray.
2016-08-22 | E-GEOD-80983 | biostudies-arrayexpress
Project description:Reconstitution of female germ-cell development in vitro is a key challenge in reproductive biology and medicine. We show here that female (XX) embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) in mice are induced into primordial germ cell-like cells (PGCLCs), which, when aggregated with female gonadal somatic cells as reconstituted ovaries, develop pre-meiotic germ cell characteristics, including X-reactivation, imprint erasure, and cyst formation. Upon transplantation under ovarian bursa, PGCLCs in the reconstituted ovaries mature into germinal vesicle-stage oocytes, which, through in vitro maturation and fertilization, contribute to fertile offspring. Our culture system serves as a robust foundation for the investigation of key properties of female germ cells, including the acquisition of totipotency, and for the reconstitution of whole female germ-cell development in vitro. We performed expression analysis of female PGC-like cells [PGCLCs; day 3 (d3), day 6 (d6), and day 3 followed by day 6 in aggregation with female embryonic day (E) 12.5 gonadal somatic cells (d3ag6)] in comparison to in vivo embryonic PGCs (E12.5 female PGCs and E9.5 PGCs) and male d6 PGCLCs. PGCLCs were marked with fluorescence of Blimp1-mVenus, stella-ECFP transgenic reporters (BVSC). PGCs were marked with fluorescence of the stella-EGFP transgenic reporter.
2012-11-05 | E-GEOD-40716 | biostudies-arrayexpress
Project description:The successful segregation of germ cells from somatic lineages is vital for sexual reproduction and species survival. In the mouse, primordial germ cells (PGCs), precursors of all germ cells, are induced from the post-implantation epiblast. Induction requires BMP4 signalling to prospective PGCs and the intrinsic action of PGC transcription factors (TFs). However, the molecular mechanisms connecting BMP4 action to induction of PGC TFs that are responsible for segregation of PGCs from somatic lineages are unknown. Here we show that the transcription factor OTX2 is a key regulator of these processes. Down-regulation of Otx2 precedes the initiation of the PGC programme both in vitro and in vivo. Deletion of Otx2 in vitro dramatically increases PGCLC differentiation efficiency and prolongs the period of PGC competence. In the absence of Otx2 activity, PGCLC differentiation becomes independent of the otherwise essential cytokine signals, with germline entry initiating even in the absence of the PGC TF Blimp1. Deletion of Otx2 in vivo increases PGC numbers. These data demonstrate that Otx2 acts repressively upstream of PGC TFs and functions as a roadblock to prevent the untimely entry of pluripotent stem cells into the PGC lineage, thereby ensuring correct spatio-temporal segregation of the germline and soma.
2018-07-06 | GSE116640 | GEO
Project description:Mammalian germ cells first establish as primordial germ cells (PGCs). There is an increasing demand to investigate the characteristics of human germ cells including PGCs. Recent studies have established the methodologies to derive human germ cells such as PGCs from human induced pluripotent cells (iPSCs). The in vitro-derived PGCs are referred to as PGC-like cells (PGCLCs) and exhibit the characteristics of the PGCs immediately after specification. Here we performed bulk RNA-sequencing (RNA-Seq) of PGCLCs and their parental iPSCs to characterize the expression signatures of human genes and transposable elements in PGCLCs in detail.
2022-03-08 | GSE167570 | GEO
Project description:The germ cell lineage ensures reproduction and heredity in metazoans. Primordial germ cells (PGCs) in mice are induced in pluripotent epiblast cells by BMP4 and WNT3, yet their mechanism of action remains elusive. Here, using in vitro PGC specification system, we show that WNT3, but not BMP4, induces many transcription factors associated with mesoderm in epiblast-like cells (EpiLCs) through beta-CATENIN. Among these, T (BRACHYURY), a classical and conserved mesodermal factor, was essential for robust activation of Blimp1 and Prdm14, two of the germline determinants. T, but not SMAD1 or beta-CATENIN/TCF1, binds distinct regulatory elements of both Blimp1 and Prdm14, and directly up-regulates these genes without BMP4 and WNT3. Without BMP4, a program induced by WNT3 prevents T from activating Blimp1 and Prdm14, demonstrating that BMP4 is permissive for PGC specification. These findings establish a fundamental role of a mesodermal gene in PGC specification, a potentially evolutionarily conserved mechanism across metazoans.
2013-12-11 | GSE49689 | GEO