Bone Morphogenetic Protein and Retinoic Acid Synergistically Specify Female Germ Cell Fate in Mice
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE94136 | GEO | 2017/09/12
SECONDARY ACCESSION(S): PRJNA369039
REPOSITORIES: GEO
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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: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:In mammals, sex differentiation of primordial germ cells (PGCs) is determined by extrinsic cues from the environment1. In female PGCs, expression of Stimulated by retinoic acid 8 (Stra8) and meiosis are induced in response to retinoic acid (RA) provided by the mesonephroi2-4. Given the widespread role of RA signaling during development8,9, the molecular mechanism specifying the competence of PGCs to timely express Stra8 and enter meiosis are unknown2,10. Here we identify gene dosage dependent roles in PGC development for Ring1 and Rnf2, two central components of the Polycomb Repressive Complex 1 (PRC1)11,13. Both paralogs are essential for PGC development between day 10.5 and 11.5 of gestation. Rnf2 is subsequently required in female PGCs for maintaining high levels of Oct4 and Nanog expression6, and for preventing premature induction of meiotic gene expression and entry into meiotic prophase. Chemical inhibition of RA signaling partially suppresses precocious Oct4 down-regulation and Stra8 activation in Rnf2-deficient female PGCs. Chromatin immunoprecipitation analyses show that Stra8 is a direct target of PRC1 and PRC2 in PGCs. These data demonstrate the importance of PRC1 gene dosage in PGC development and in coordinating the timing of sex differentiation of female PGCs by antagonizing extrinsic RA signaling. Gene expression of mouse primordial germ cells was analysed using RNAseq method. Primodial germ cells were purified from embryos carrying Oct4(-delta-PE)-GFP transgene by FACS.
2013-03-14 | E-GEOD-42852 | biostudies-arrayexpress
Project description:In mammals, sex differentiation of primordial germ cells (PGCs) is determined by extrinsic cues from the environment1. In female PGCs, expression of Stimulated by retinoic acid 8 (Stra8) and meiosis are induced in response to retinoic acid (RA) provided by the mesonephroi2-4. Given the widespread role of RA signaling during development8,9, the molecular mechanism specifying the competence of PGCs to timely express Stra8 and enter meiosis are unknown2,10. Here we identify gene dosage dependent roles in PGC development for Ring1 and Rnf2, two central components of the Polycomb Repressive Complex 1 (PRC1)11,13. Both paralogs are essential for PGC development between day 10.5 and 11.5 of gestation. Rnf2 is subsequently required in female PGCs for maintaining high levels of Oct4 and Nanog expression6, and for preventing premature induction of meiotic gene expression and entry into meiotic prophase. Chemical inhibition of RA signaling partially suppresses precocious Oct4 down-regulation and Stra8 activation in Rnf2-deficient female PGCs. Chromatin immunoprecipitation analyses show that Stra8 is a direct target of PRC1 and PRC2 in PGCs. These data demonstrate the importance of PRC1 gene dosage in PGC development and in coordinating the timing of sex differentiation of female PGCs by antagonizing extrinsic RA signaling. Gene expression of mouse primordial germ cells was analysed using Affymetrix microarray. Primodial germ cells were purified from embryos carrying Oct4(deltaPE)-GFP transgene by FACS.
2013-03-14 | E-GEOD-42782 | biostudies-arrayexpress
Project description:The DND microRNA-mediated repression inhibitor 1 (DND1) is a conserved RNA binding protein (RBP) and plays an important role in survival and maintenance of primordial germ cells (PGCs) and the development of the male germline in zebrafish and mice. It was shown to be expressed in human pluripotent stem cells (PSCs), PGCs, and spermatogonia, but little is known about its specific role in pluripotency and human germline development. Here we use CRISPR/Cas mediated knockout and PGC-like cell (PGCLC) differentiation in human iPSCs to analyse if DND1 (1) plays a role in maintaining pluripotency and (2) in specification of PGCLCs. We generated several clonal lines with biallelic loss of function mutations and analysed their potential to differentiate towards PGCLCs and their gene expression on RNA and protein level via bulk RNA sequencing and mass spectrometry. The generated knockout iPSCs showed no differences in pluripotency gene expression, proliferation nor trilineage differentiation potential, but yielded reduced numbers o PGCLCs compared to their parental iPSCs. RNAseq analysis in PGCLCs showed significantly reduced expression of genes associated with cellular developmental processes and cell differentiation in knockout cells, including known markers for PGCs (NANOS3, SOX17, PRDM1, EPCAM) and naïve pluripotency (TFCP2L, DNMT3L).
2021-12-10 | PXD023802 | Pride
Project description:We re-analyzed gene expression in the primordial germ cell (PGC) specification pathway in vitro, by using previously deposited microarray data. The germ cell lineage produces either spermatozoa or oocytes and, by their fusion, creates zygotes with full developmental potential, thereby perpetuating and diversifying organisms' genetic as well as epigenetic information across generations. In mice, PGCs are specified in the most posterior part of epiblast (monolayer epitherium-like cells, from which the whole embryonic part of conceptus will be derived) around embryonic day (E) 6.25 by a cytokine BMP4. To recaptulate this pathway, an in vitor system was established by Hayashi et al. (2011, 2012): epiblast-like cells (EpiLCs) are induced from embryonic stem cells (ESCs) by cytokine Activin and bFGF, and then PGC-like cells (PGCLCs) are induced from EpiLC by cytokines including BMP4. Similar to the in vivo PGCs, PGCLCs exhibit mesoderm-like profile 2 days after induction (d2 PGCLCs), and then 6 days after induction (d6 PGCLCs), they acquire profiles like PGCs at E9.5 in vivo.
2015-03-20 | GSE60018 | GEO
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:Avian sex is determined by various factors, such as the dosage of DMRT1 and cell-autonomous mechanisms. While the sex-determination mechanism in gonads is well analyzed, the mechanism in germ cells remains unclear. In this study, we explored the gene expression profiles of male and female primordial germ cells (PGCs) during embryogenesis in chickens to predict the mechanism of sex-determination. Male and female PGCs were isolated from blood and gonads with a purity > 96% using flow cytometry and analyzed using RNA-seq. Prior to settlement in the gonads, female circulating PGCs (cPGCs) obtained from blood displayed sex-biased expression. Gonadal PGCs (gPGCs) also displayed sex-biased expression, and the number of female-biased genes detected was higher than male-biased genes. The female-biased genes in gPGCs were enriched in some metabolic processes. To reveal the mechanisms underlying the transcriptional regulation of female-biased genes in gPGCs, we performed stimulation tests. Stimulation with retinoic acid against cultured gPGCs derived from male embryos resulted in the upregulation of several female-biased genes. Overall, our results suggest that sex determination of avian PGCs possess aspects of both cell-autonomous and somatic cell regulation. Moreover, it appears that sex determination occurs earlier in females than in males.
2022-02-24 | GSE188689 | GEO
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:Nascent human primordial germ cell-like cells (PGCLCs) can be specified with BMP2 or BMP4 together with SCF and EGF from germline competent embryonic stem cells which are maintained in the presence of inhibitors for GSK3, MEK, p38 and JNK together with FGF2, TGFβ and LIF (4i ESC). Here we discovered that Activin A and retinoic acid treatment after PGC specification induced genes expressed in migrating and gonadal PGCs in vivo, including DMRT1 and CDH5. Ectopic expression of DMRT1 and SOX17 resulted in repression of pluripotent genes as well as induction of DAZL and a subset of mitotic arrest PGC genes. Our findings provide novel insights on the roles of DMRT1 for the transition from nascent PGCs towards germline determination and molecular mechanisms of early human germline development.
2023-07-18 | GSE223035 | GEO