An RNA-interacting protein network regulates pluripotency in mammalian primordial germ cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Niels Geijsen
PROVIDER: E-GEOD-39547 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:Primordial germ cells (PGCs) are the embryonic precursors to egg and sperm. When removed from the embryonic gonad, PGCs can give rise to embryonic germ cell lines (EGs), pluripotent stem cells that display most of the characteristics of embryonic stem cells (ESCs) including the ability to form teratomas and to contribute to chimera formation. In mice, EG cells can be derived between E8.5 up to E12.5 of embryonic development, at which point the PGCs undergo sexual differentiation and in the male transition into unipotent gonocytes. Dazl, a germ cell-specific RNA-binding protein, is specifically expressed in developing PGCs and is required for proper germ cell development. Dazl knockout mice are infertile, but the molecular mechanisms underlying this phenotype are still unknown. Here we demonstrate that Dazl localizes in granular structures in mammalian PGCs but not in ESCs. We demonstrate Dazl plays a central role in a large mRNA/protein interactive network that includes members of Fragile-X family RNA-binding proteins. We demonstrate that Dazl and Fxr1 play a central role in these granules and directly regulate the translation of specific core pluripotency factors, including Sox2 and Suz12. Global gene expression changes during in vitro germ cell differentiation from murine ES cells and comparison to in vivo germ cells. In vitro primordial germ cells differentiated from Stella-GFP ES cells and Dazl-GFP ES cells. The GFP-positive cells were isolated by FACS analysis. Dazl-GFP+ cells were isolated from transgenic E13.5 embryonic gonads and P7-P11 juvenile mouse testes. The global gene expression profiles were analyzed by Agilent Mouse Whole Genome 4X44K one-color microarrays. Two replicates per condition.
2013-12-31 | E-GEOD-39546 | biostudies-arrayexpress
Project description:Primordial germ cells (PGCs) are the embryonic precursors to egg and sperm. When removed from the embryonic gonad, PGCs can give rise to embryonic germ cell lines (EGs), pluripotent stem cells that display most of the characteristics of embryonic stem cells (ESCs) including the ability to form teratomas and to contribute to chimera formation. In mice, EG cells can be derived between E8.5 up to E12.5 of embryonic development, at which point the PGCs undergo sexual differentiation and in the male transition into unipotent gonocytes. Dazl, a germ cell-specific RNA-binding protein, is specifically expressed in developing PGCs and is required for proper germ cell development. Dazl knockout mice are infertile, but the molecular mechanisms underlying this phenotype are still unknown. Here we demonstrate that Dazl localizes in granular structures in mammalian PGCs but not in ESCs. We demonstrate Dazl plays a central role in a large mRNA/protein interactive network that includes members of Fragile-X family RNA-binding proteins. We demonstrate that Dazl and Fxr1 play a central role in these granules and directly regulate the translation of specific core pluripotency factors, including Sox2 and Suz12. RNA species interacting specifically with Dazl in primordial germ cells were identified by RNA-IP microarray analysis. This dataset contains data from native RNA-IPs without UV-crosslinking. Two independent native RNA-IP (anti-V5 and anti-GFP) experiments from in vitro derived primordial germ cells expressing Dazl-GFP-V5. Input Total RNA and mock IP using normal mouse IgG were used as controls. Control and IP-enriched RNA samples were analyzed by Agilent Mouse Whole Genome 4X44K one-color microarrays.
2013-12-31 | E-GEOD-39544 | biostudies-arrayexpress
Project description:Primordial germ cells (PGCs) are the embryonic precursors to egg and sperm. When removed from the embryonic gonad, PGCs can give rise to embryonic germ cell lines (EGs), pluripotent stem cells that display most of the characteristics of embryonic stem cells (ESCs) including the ability to form teratomas and to contribute to chimera formation. In mice, EG cells can be derived between E8.5 up to E12.5 of embryonic development, at which point the PGCs undergo sexual differentiation and in the male transition into unipotent gonocytes. Dazl, a germ cell-specific RNA-binding protein, is specifically expressed in developing PGCs and is required for proper germ cell development. Dazl knockout mice are infertile, but the molecular mechanisms underlying this phenotype are still unknown. Here we demonstrate that Dazl localizes in granular structures in mammalian PGCs but not in ESCs. We demonstrate Dazl plays a central role in a large mRNA/protein interactive network that includes members of Fragile-X family RNA-binding proteins. We demonstrate that Dazl and Fxr1 play a central role in these granules and directly regulate the translation of specific core pluripotency factors, including Sox2 and Suz12. Global gene expression changes following Dazl knockdown in in vitro primordial germ cells. In vitro primordial germ cells carrying control and Dazl knockdown shRNAs were generated from Oct4-GFP ES cells and isolated by FACS analysis. The global gene expression profiles were analyzed by Agilent Mouse Whole Genome 4X44K one-color microarrays.
2013-12-31 | E-GEOD-39535 | biostudies-arrayexpress
Project description:Primordial germ cells (PGCs) are the embryonic precursors to egg and sperm. When removed from the embryonic gonad, PGCs can give rise to embryonic germ cell lines (EGs), pluripotent stem cells that display most of the characteristics of embryonic stem cells (ESCs) including the ability to form teratomas and to contribute to chimera formation. In mice, EG cells can be derived between E8.5 up to E12.5 of embryonic development, at which point the PGCs undergo sexual differentiation and in the male transition into unipotent gonocytes. Dazl, a germ cell-specific RNA-binding protein, is specifically expressed in developing PGCs and is required for proper germ cell development. Dazl knockout mice are infertile, but the molecular mechanisms underlying this phenotype are still unknown. Here we demonstrate that Dazl localizes in granular structures in mammalian PGCs but not in ESCs. We demonstrate Dazl plays a central role in a large mRNA/protein interactive network that includes members of Fragile-X family RNA-binding proteins. We demonstrate that Dazl and Fxr1 play a central role in these granules and directly regulate the translation of specific core pluripotency factors, including Sox2 and Suz12. RNA species interacting specifically with Dazl in primordial germ cells were identified by RNA-IP microarray analysis. This dataset contains data from UV-crosslinked RNA-IPs from in vitro PGC lysates. UV-crosslinked RNA-IP (anti-GFP and anti-PABP1) experiments from in vitro derived primordial germ cells expressing Dazl-GFP-V5. Input Total RNA and polyA-binding protein 1 (PABP1) IP were used as controls. Control and IP-enriched RNA samples were analyzed by Agilent Mouse Whole Genome 4X44K one-color microarrays. Two replicates for each condition were done.
2013-12-31 | E-GEOD-39541 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series
2015-07-01 | E-GEOD-69357 | biostudies-arrayexpress
Project description:Embryonic stem cell (ESC) cultures display a heterogeneous gene expression profile, ranging from a pristine naïve pluripotent state to a primed epiblast state. While it is known that the addition of inhibitors of GSK3β and MEK (so-called 2i conditions) push ESC cultures towards a more homogeneous naïve pluripotent state, the molecular underpinnings of this naïve transition are not completely understood. Here we demonstrate that Dazl, a RNA-binding protein previously thought to be expressed specifically in developing primordial germ cells (PGCs), marks a subpopulation of ESCs in vitro that is actively transitioning toward naïve pluripotency. In the absence of Dazl expression, ESCs fail to induce proper expression of Tet enzymes required for 5-hydroxymethylation in 2i-culture conditions. As a result, 5-hydroxymethylation of methylated cystosine residues is impaired. Indeed, we demonstrate that Tet1 and Tet2 are mRNA targets of Dazl, indicating that Dazl might play a role in protection or stabilizing these mRNA molecules. Our results provide insight in the regulation of the acquisition of naïve pluripotency and demonstrate that Dazl is required for TET-mediated cytosine hydroxymethylation in cells that are actively reprogramming to a pluripotent ground state. RNA-IP experiments were used to identify the RNA species bound to DAZL.
2015-07-01 | E-GEOD-69356 | biostudies-arrayexpress
Project description:Embryonic stem cell (ESC) cultures display a heterogeneous gene expression profile, ranging from a pristine naïve pluripotent state to a primed epiblast state. While it is known that the addition of inhibitors of GSK3? and MEK (so-called 2i conditions) push ESC cultures towards a more homogeneous naïve pluripotent state, the molecular underpinnings of this naïve transition are not completely understood. Here we demonstrate that Dazl, a RNA-binding protein previously thought to be expressed specifically in developing primordial germ cells (PGCs), marks a subpopulation of ESCs in vitro that is actively transitioning toward naïve pluripotency. In the absence of Dazl expression, ESCs fail to induce proper expression of Tet enzymes required for 5-hydroxymethylation in 2i-culture conditions. As a result, 5-hydroxymethylation of methylated cystosine residues is impaired. Indeed, we demonstrate that Tet1 and Tet2 are mRNA targets of Dazl, indicating that Dazl might play a role in protection or stabilizing these mRNA molecules. Our results provide insight in the regulation of the acquisition of naïve pluripotency and demonstrate that Dazl is required for TET-mediated cytosine hydroxymethylation in cells that are actively reprogramming to a pluripotent ground state. Two independent mouse ES cell lines, Dazl-GFP and Stella-GFP, were cultured on ?-irradiated feeder MEFs in DMEM containing 15% FBS or serum-free B27N2 medium both supplemented with leukemia inhibitory factor (LIF) (Ying 2008). For the 2i experiments, 1µM MEK inhibitor PD0325901 (Axon Medchem), and 5 µM GSK3? inhibitor Kenpaullone (Tocris), were used. Cells were harvest at 0, 3 and 10 days in each condition for expression microarray analysis.
2015-07-01 | E-GEOD-69055 | biostudies-arrayexpress
Project description:Induced overexpression of Pdx1 in activin-induced endoderm population resulted in the upregulation of pancreas-related genes such as insulin 1 and 2 at day 20. To enhance the developmental progression from the pancreatic bud to the formation of the endocrine lineages, we next expressed neurogenin3 (Ngn3) together with Pdx-1. Induced overexpression of Pdx1 together with Ngn3 dramatically increased Insulin 1 mRNA by day 9 differentiation. The levels of insulin 1 mRNA present in the induced EBs represented approximately 100 % of that found in insulinoma cell line, betaTC6. We also confirmed insulin and C-peptide staining by immunohistochemistry. These cells process and secrete insulin and respond to various insulin secretagogues. These inductive effects were restricted to c-kit+ endoderm enriched EB-derived populations suggesting that Pdx1/Ngn3 functions at the level of pancreatic specification of endoderm in this model. Microarray analysis showed that Pdx1/Ngn3 regulated the expression of a broad spectrum of pancreatic endocrine cell-related genes. On day 4 of a multiday differentiation protocol, EBs were dissociated and cultured in serum-free growth medium supplemented with differentiation facots, with or without Dox (1 ug/ml). On day 6, EBs were replated on gelatin in 12-well low-cluster dishes (Nunc) to obtain non-adherent floating EBs with or without Dox (1 ug/ml) and cultured out to day 13, at which time RNA was harvested for microarray analysis.
2010-08-24 | E-GEOD-23752 | biostudies-arrayexpress
Project description:Chromatin modifications have been implicated in the self-renewal and differentiation of embryonic stem cells (ESCs). However, the function of histone variant H2A.Z in ESCs remains unclear. We show that H2A.Z is highly enriched at promoters and enhancers and is required for both efficient self-renewal and differentiation of murine ESCs. H2A.Z deposition leads to an abnormal nucleosome structure, decreased nucleosome occupancy and increased chromatin accessibility. In self-renewing ESCs, knockdown of H2A.Z compromises OCT4 binding to its target genes and leads to decreased binding of MLL complexes to active genes and of PRC2 complex to repressed genes in self-renewal of ESCs. During differentiation of ESCs, inhibition of H2A.Z also compromises RA-induced RARα binding, activation of differentiation markers and the repression of pluripotency genes. We propose that H2A.Z mediates such contrasting activities by acting as a 'general facilitator' that generates access for a variety of complexes both activating and repressive. ChIP-Seq in murine embryonic stem (mES) cells for H2A.Z and acetylated H2A.Z. ChIP-Seq of H3K4me3, H3K27me3, RbBP5, SUZ12 and OCT4 for mES cells of both H2A.Z RNAi knockdown and shLuc control. ChIP-Seq of RARalpha in H2A.Z knockdown (withdraw of LIF and exposure to RA for 3h) and control cells. MNase-Seq and chromatin accessibility assay using Benzonase digestion followed by next-generation sequencing for mES cells of both H2A.Z RNAi knockdown and shLuc control. ChIP-Seq of H2A.Z and H3K4me3 for mES cells of both MLL4 RNAi knockdown and shLuc control. RNA-Seq for mES cells of H2A.Z knockdown and shluc control. RNA-Seq for embryonic bodies derived from mES cells (H2A.Z knockdown and shLuc control) at day 3 and day 7.
2012-12-20 | E-GEOD-34483 | biostudies-arrayexpress
Project description:Cardiac disease accounts for the largest proportion of adult mortality and morbidity in the industrialized world. However, progress toward improved clinical treatments is hampered by an incomplete understanding of the genetic programs controlling early cardiogenesis. To better understand this process, we set out to identify genes whose expression is enriched within early cardiac fated populations, obtaining the transcriptional signatures of mouse embryonic stem cells (mESCs) differentiating along a cardiac path. We compared the RNA profiles of cardiac precursors cells (CPCs) with time-matched non-CPCs and undifferentiated mESCs, using a transgenic mESC line harboring an Nkx2-5 cardiac-specific regulatory sequence driving green fluorescent protein (GFP) to facilitate selection of CPCs. Approximately 24% (43/176) of the transcripts enriched in the CPC population have known roles in cardiac function or development. Importantly, we evaluated the biological relevance of a subset 31/133 (23%) of the remaining candidate genes by in situ hybridization and report that all were expressed in key cardiac structures during cardiogenesis (embryonic day, E7.5 - 9.5), many of which were previously uncharacterized. These data demonstrate the power of mESC differentiation to model specific developmental processes and provide a valuable resource that may be mined to further elucidate the genetic programs underlying cardiogenesis. Experiment Overall Design: Transgenic mouse embryonic stem cell line (Nkx2-5-GFP, PMID:9834187) contains an Nkx2-5 cardiac-specific transcription factor driving expression of green fluorescent protein (GFP). Cells were differentiated through embryoid body formation (hanging droplet technique) as described in PMID:8155574. Following primary mouse embryonic fibroblasts (PMEF) feeder subtraction, mESCs were dissociated and resuspended in differentiation medium. Cells at this time point were designated as day 0. Following incubation for two days, the EBs were transferred, in suspension, to poly-HEMA coated tissue culture dishes to prevent cell attachment and grown in medium containing ascorbic acid PMID:12668514) which was replenished every 3 days thereafter. Experiment Overall Design: Cells were harvested on days 0, 4 and 6 in three biological replicates Experiment Overall Design: each (1a, 2a and 3a), washed in PBS for 30 minutes at 37ºC then resuspended in 0.25% trypsin for 7 minutes. Experiment Overall Design: Cells from day 6 were then sorted using the BD Biosciences FACSAria⢠Cell sorting system into GFP+ and GFP- fractions into RLT buffer (RNeasy, Qiagen). This sorting separates cells into two groups reflecting their differentiation into those fated to a non-cardiac lineage (GFP negative) and to a cardiac lineage (GFP positive).
2009-03-15 | E-GEOD-10970 | biostudies-arrayexpress