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An RNA-interacting protein network regulates pluripotency in mammalian primordial germ cells (Dazl knockdown)

ABSTRACT: 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.

ORGANISM(S): Mus musculus

SUBMITTER: Niels Geijsen

PROVIDER: E-GEOD-39535 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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An RNA-interacting protein network regulates pluripotency in mammalian primordial germ cells (Dazl native RNA-IP)

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

An RNA-interacting protein network regulates pluripotency in mammalian primordial germ cells (Dazl UV RNA-IP)

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.

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An RNA-interacting protein network regulates pluripotency in mammalian primordial germ cells (in vitro, in vivo)

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

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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 juvenile testicular germ cells were identified by RNA-IP microarray analysis. This dataset contains data from native RNA-IPs from P14 Dazl-GFP transgenic mouse testes. Native RNA-IP (anti-V5 and anti-PABP1) experiments from juvenile testicular germ cells expressing Dazl-GFP-V5. Input Total RNA and mock IP with normal mouse IgG 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-39545 | biostudies-arrayexpress

Dazl is required for TET-mediated reprogramming to a naïve pluripotent state (II)

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

An RNA-interacting protein network regulates pluripotency in mammalian primordial germ cells (Dazl knockdown)

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.

2013-12-31 | GSE39535 | GEO

An RNA-interacting protein network regulates pluripotency in mammalian primordial germ cells (Dazl native RNA-IP)

2013-12-31 | GSE39544 | GEO

An RNA-interacting protein network regulates pluripotency in mammalian primordial germ cells (Dazl UV RNA-IP)

2013-12-31 | GSE39541 | GEO

Dazl is required for TET-mediated reprogramming to a naïve pluripotent state (I)

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

An RNA-interacting protein network regulates pluripotency in mammalian primordial germ cells (in vitro, in vivo)

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.

2013-12-31 | GSE39546 | GEO

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