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Claire Chazaud - One of the best experts on this subject based on the ideXlab platform.
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Primitive Endoderm Differentiation: From Specification to Epithelialization.
Current Topics in Developmental Biology, 2018Co-Authors: Cécilia Bassalert, Lorena Valverde-estrella, Claire ChazaudAbstract:At the time of implantation, the mouse blastocyst has developed three cell lineages: the epiblast (Epi), the Primitive Endoderm (PrE), and the trophectoderm (TE). The PrE and TE are extraembryonic tissues but their interactions with the Epi are critical to sustain embryonic growth, as well as to pattern the embryo. We review here the cellular and molecular events that lead to the production of PrE and Epi lineages and discuss the different hypotheses that are proposed for the induction of these cell types. In the second part, we report the current knowledge about the epithelialization of the PrE.
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Primitive Endoderm differentiation: from specification to epithelium formation
Philosophical Transactions of the Royal Society B: Biological Sciences, 2014Co-Authors: Stéphanie Hermitte, Claire ChazaudAbstract:In amniotes, Primitive Endoderm (PrE) plays important roles not only for nutrient support but also as an inductive tissue required for embryo patterning. PrE is an epithelial monolayer that is visible shortly before embryo implantation and is one of the first three cell lineages produced by the embryo. We review here the molecular mechanisms that have been uncovered during the past 10 years on PrE and epiblast cell lineage specification within the inner cell mass of the blastocyst and on their subsequent steps of differentiation.
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A close look at the mammalian blastocyst: epiblast and Primitive Endoderm formation.
Cellular and molecular life sciences : CMLS, 2014Co-Authors: Jérôme Artus, Claire ChazaudAbstract:During early development, the mammalian embryo undergoes a series of profound changes that lead to the formation of two extraembryonic tissues—the trophectoderm and the Primitive Endoderm. These tissues encapsulate the pluripotent epiblast at the time of implantation. The current model proposes that the formation of these lineages results from two consecutive binary cell fate decisions. The first controls the formation of the trophectoderm and the inner cell mass, and the second controls the formation of the Primitive Endoderm and the epiblast within the inner cell mass. While early mammalian embryos develop with extensive plasticity, the embryonic pattern prior to implantation is remarkably reproducible. Here, we review the molecular mechanisms driving the cell fate decision between Primitive Endoderm and epiblast in the mouse embryo and integrate data from recent studies into the current model of the molecular network regulating the segregation between these lineages and their subsequent differentiation.
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Primitive Endoderm differentiates via a three step mechanism involving nanog and rtk signaling
Developmental Cell, 2011Co-Authors: Stephen Frankenberg, Francois Gerbe, Sylvain Bessonnard, Corinne Belville, Pierre Pouchin, Olivier Bardot, Claire ChazaudAbstract:Summary During preimplantation mouse development, the inner cell mass (ICM) differentiates into two cell lineages—the epiblast and the Primitive Endoderm (PrE)—whose precursors are identifiable by reciprocal expression of Nanog and Gata6, respectively. PrE formation depends on Nanog by a non-cell-autonomous mechanism. To decipher early cell- and non-cell-autonomous effects, we performed a mosaic knockdown of Nanog and found that this is sufficient to induce a PrE fate cell autonomously. Strikingly, in Nanog null embryos, Gata6 expression is maintained, showing that initiation of the PrE program is Nanog independent. Treatment of Nanog null embryos with pharmacological inhibitors revealed that RTK dependency of Gata6 expression is initially direct but later indirect via Nanog repression. Moreover, we found that subsequent expression of Sox17 and Gata4—later markers of the PrE—depends on the presence of Fgf4 produced by Nanog-expressing cells. Thus, our results reveal three distinct phases in the PrE differentiation program.
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The RNA-Binding Protein Unr Prevents Mouse Embryonic Stem Cells Differentiation Toward the Primitive Endoderm Lineage
STEM CELLS, 2011Co-Authors: Habiba Elatmani, Claire Chazaud, Virginie Dormoy-raclet, Pierre Dubus, François Dautry, Hélène Jacquemin-sablonAbstract:The maintenance of embryonic stem cells (ESCs) pluripotency depends on key transcription factors, chromatin remodeling proteins, and microRNAs. The roles of RNA-binding proteins are however poorly understood. We report that the cytoplasmic RNA-binding protein Unr prevents the differentiation of ESCs into Primitive Endoderm (PrE). We show that unr knockout (unr(-/-) ) ESCs spontaneously differentiate into PrE, and that Unr re-expression in unr(-/-) ESCs reverses this phenotype. Nevertheless, unr(-/-) ESCs retain pluripotency, producing differentiated teratomas, and the differentiated unr(-/-) ESCs coexpress the PrE inducer Gata6 and the pluripotency factors Oct4, Nanog, and Sox2. Interestingly, in the differentiated unr(-/-) ESCs, Nanog and Sox2 exhibit a dual nuclear and cytoplasmic localization. This situation, that has never been reported, likely reflects an early differentiation state toward PrE. Finally, we show that Unr destabilizes Gata6 mRNAs and we propose that the post-transcriptional repression of Gata6 expression by Unr contributes to the stabilization of the ESCs pluripotent state.
Craig C. Malbon - One of the best experts on this subject based on the ideXlab platform.
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Gα13 Signals via p115RhoGEF Cascades Regulating JNK1 and Primitive Endoderm Formation
The Journal of biological chemistry, 2004Co-Authors: Yi-nan Lee, Craig C. Malbon, Hsien-yu WangAbstract:Abstract The heterotrimeric G-protein G13 mediates the formation of Primitive Endoderm from mouse P19 embryonal carcinoma cells in response to retinoic acid, signaling to the level of activation of c-Jun N-terminal kinase. The signal linkage map from MEKK1/MEKK4 to MEK1/MKK4 to JNK is obligate in this Gα13-mediated pathway, whereas that between Gα13 and MEKKs is not known. The overall pathway to Primitive Endoderm formation was shown to be inhibited by treatment with Clostridium botulinum C3 exotoxin, a specific inactivator of RhoA family members. Constitutively active Gα13 was found to activate RhoA as well as Cdc42 and Rac1 in these cells. Although constitutively active Cdc42, Rac1, and RhoA all can activate JNK1, only the RhoA mutant was able to promote formation of Primitive Endoderm, mimicking expression of the constitutively activated Gα13. Expression of the constitutively active mutant form of p115RhoGEF (guanine nucleotide exchange factor) was found to activate RhoA and JNK1 activities. Expression of the dominant negative p115RhoGEF was able to inhibit activation of both RhoA and JNK1 in response to either retinoic acid or the expression of a constitutively activated mutant of Gα13. Expression of the dominant negative mutants of RhoA as well as those of either Cdc42 or Rac1, but not Ras, attenuated Gα13-stimulated as well as retinoic acid-stimulated activation of all three of these small molecular weight GTPases, suggesting complex interrelationships among the three GTPases in this pathway. The formation of Primitive Endoderm in response to retinoic acid also could be blocked by expression of dominant negative mutants of RhoA, Cdc42, or Rac1. Thus, the signal propagated from Gα13 to JNK requires activation of p115RhoGEF cascades, including p115RhoGEF itself, RhoA, Cdc42, and Rac1. In a concerted effort, RhoA in tandem with Cdc42 and Rac1 activates the MEKK1/4, MEK1/MKK4, and JNK cascade, thereby stimulating formation of Primitive Endoderm.
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activation of the β catenin lef tcf pathway is obligate for formation of Primitive Endoderm by mouse f9 totipotent teratocarcinoma cells in response to retinoic acid
Journal of Biological Chemistry, 2002Co-Authors: Tong Liu, Yi-nan Lee, Craig C. Malbon, Hsien-yu WangAbstract:Next Section Abstract The morphogen retinoic acid promotes the formation of Primitive Endoderm in mouse F9 teratocarcinoma cells as does the stimulation of the Frizzled-1 pathway. We investigated whether the β-catenin/Lef-Tcf-sensitive transcriptional pathway activated by Frizzled-1 plays a role in the retinoic acid-induced pathway to Primitive Endoderm formation. An analysis of Lef-Tcf-sensitive transcription reveals increased transcription at 1 and 4 h post-treatment with retinoic acid. The stimulation of Lef-Tcf-sensitive transcription as well as the formation of Primitive Endoderm was accompanied by the stabilization of β-catenin as observed in activation of the Frizzled-1 pathway. Transient transfection of F9 cells with an expression vector harboring a dominant-negative mutant of Tcf4 resulted in the attenuation of both the increase in Lef-Tcf-sensitive transcription and formation of Primitive Endoderm in response to the morphogen. Clones stably transfected to express the dominant-negative Tcf4 displayed a block in retinoic acid-induced activation of Lef-Tcf-sensitive transcription and Primitive Endoderm formation. These data reveal the obligate role of the β-catenin/Lef-Tcf transcriptional pathway in the action of the morphogen retinoic acid.
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Activation of the β-catenin/Lef-Tcf pathway is obligate for formation of Primitive Endoderm by mouse F9 totipotent teratocarcinoma cells in response to retinoic acid
The Journal of biological chemistry, 2002Co-Authors: Tong Liu, Yi-nan Lee, Craig C. Malbon, Hsien-yu WangAbstract:Next Section Abstract The morphogen retinoic acid promotes the formation of Primitive Endoderm in mouse F9 teratocarcinoma cells as does the stimulation of the Frizzled-1 pathway. We investigated whether the β-catenin/Lef-Tcf-sensitive transcriptional pathway activated by Frizzled-1 plays a role in the retinoic acid-induced pathway to Primitive Endoderm formation. An analysis of Lef-Tcf-sensitive transcription reveals increased transcription at 1 and 4 h post-treatment with retinoic acid. The stimulation of Lef-Tcf-sensitive transcription as well as the formation of Primitive Endoderm was accompanied by the stabilization of β-catenin as observed in activation of the Frizzled-1 pathway. Transient transfection of F9 cells with an expression vector harboring a dominant-negative mutant of Tcf4 resulted in the attenuation of both the increase in Lef-Tcf-sensitive transcription and formation of Primitive Endoderm in response to the morphogen. Clones stably transfected to express the dominant-negative Tcf4 displayed a block in retinoic acid-induced activation of Lef-Tcf-sensitive transcription and Primitive Endoderm formation. These data reveal the obligate role of the β-catenin/Lef-Tcf transcriptional pathway in the action of the morphogen retinoic acid.
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Expression of Gα13 (Q226L) Induces P19 Stem Cells to Primitive Endoderm via MEKK1, 2, or 4
The Journal of biological chemistry, 2001Co-Authors: Hsien-yu Wang, Jyotshnabala Kanungo, Craig C. MalbonAbstract:Galpha13 mediates the ability of the morphogen retinoic acid to promote Primitive Endoderm formation from mouse P19 embryonal carcinoma stem cells, a process that includes the obligate activation of Jun N-terminal kinase. Expression of the constitutively activated (Q226L) GTPase-deficient form of Galpha13 mimics retinoic acid and was used to investigate the signaling upstream of Primitive Endoderm formation. Jun N-terminal kinase 1 activity, MEK1,2, MKK4, and MEKK1 were constitutively activated in clones stably transfected to express Q226L Galpha13. Dominant negative forms of MEKK1 and MEKK4 were expressed stably in the clones harboring Q226L Galpha13. Expression of dominant negative versions of either MEKK1 or MEKK4 effectively blocks both the activation of Jun N-terminal kinase as well as the formation of Primitive Endoderm. Depletion of MEKK1, -2, or -4 by antisense oligodeoxynucleotides suppressed signaling from Q226L Galpha13 to JNK1 and Primitive Endoderm formation. We demonstrate that the signal linkage map from Galpha13 activation to Primitive Endoderm formation in these stem cells requires activation at three levels of the mitogen-activated protein kinase cascade: MEKK1, -2, or -4 for MAP kinase kinase kinase; MKK4 and/or MEK1 for MAP kinase kinase; and JNK1 for MAP kinase.
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Differentiation of F9 Teratocarcinoma Stem Cells to Primitive Endoderm Is Regulated by the Giα2/Gsα Axis via Phospholipase C and Not Adenylylcyclase
The Journal of biological chemistry, 1996Co-Authors: Ping Gao, Craig C. MalbonAbstract:Morphogen-induced decline in Gialpha triggers F9 teratocarcinoma stem cells to progress to Primitive Endoderm via activation of protein kinase C and mitogen-activated protein kinase (Gao, P., and Malbon, C. C. (1996) J. Biol. Chem. 271, 9002-9008). Constitutive expression of Gialpha2 blocks, whereas expression of Gsalpha provokes, progression to Primitive Endoderm, permitting identification of the effectors of the response-utilizing chimera created between Gialpha2 and Gsalpha. N-terminal substitution of Gsalpha with Gialpha2 sequence to create chimera Gialpha2 (1-54)/Gsalpha produced a chimera that activated adenylylcyclase but abolished progression to Primitive Endoderm and activation of phospholipase C. C-terminal substitution of Gsalpha with Gialpha2 sequence to Gsalpha/Gialpha2 (320-355) enhanced the ability of Gsalpha to promote progression. The Q205L-activated mutant of Gialpha2 suppresses, whereas the G225T-activated mutant of Gsalpha strongly activates phospholipase C and progression in these cells. The N-terminal region of Gsalpha (residues 62-86) appears to act as a dominant switch for the Gsalpha- (activation) versus Gialpha2- (suppression) mediated control of phospholipase C and progression to Primitive Endoderm.
Hsien-yu Wang - One of the best experts on this subject based on the ideXlab platform.
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Gα13 Signals via p115RhoGEF Cascades Regulating JNK1 and Primitive Endoderm Formation
The Journal of biological chemistry, 2004Co-Authors: Yi-nan Lee, Craig C. Malbon, Hsien-yu WangAbstract:Abstract The heterotrimeric G-protein G13 mediates the formation of Primitive Endoderm from mouse P19 embryonal carcinoma cells in response to retinoic acid, signaling to the level of activation of c-Jun N-terminal kinase. The signal linkage map from MEKK1/MEKK4 to MEK1/MKK4 to JNK is obligate in this Gα13-mediated pathway, whereas that between Gα13 and MEKKs is not known. The overall pathway to Primitive Endoderm formation was shown to be inhibited by treatment with Clostridium botulinum C3 exotoxin, a specific inactivator of RhoA family members. Constitutively active Gα13 was found to activate RhoA as well as Cdc42 and Rac1 in these cells. Although constitutively active Cdc42, Rac1, and RhoA all can activate JNK1, only the RhoA mutant was able to promote formation of Primitive Endoderm, mimicking expression of the constitutively activated Gα13. Expression of the constitutively active mutant form of p115RhoGEF (guanine nucleotide exchange factor) was found to activate RhoA and JNK1 activities. Expression of the dominant negative p115RhoGEF was able to inhibit activation of both RhoA and JNK1 in response to either retinoic acid or the expression of a constitutively activated mutant of Gα13. Expression of the dominant negative mutants of RhoA as well as those of either Cdc42 or Rac1, but not Ras, attenuated Gα13-stimulated as well as retinoic acid-stimulated activation of all three of these small molecular weight GTPases, suggesting complex interrelationships among the three GTPases in this pathway. The formation of Primitive Endoderm in response to retinoic acid also could be blocked by expression of dominant negative mutants of RhoA, Cdc42, or Rac1. Thus, the signal propagated from Gα13 to JNK requires activation of p115RhoGEF cascades, including p115RhoGEF itself, RhoA, Cdc42, and Rac1. In a concerted effort, RhoA in tandem with Cdc42 and Rac1 activates the MEKK1/4, MEK1/MKK4, and JNK cascade, thereby stimulating formation of Primitive Endoderm.
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activation of the β catenin lef tcf pathway is obligate for formation of Primitive Endoderm by mouse f9 totipotent teratocarcinoma cells in response to retinoic acid
Journal of Biological Chemistry, 2002Co-Authors: Tong Liu, Yi-nan Lee, Craig C. Malbon, Hsien-yu WangAbstract:Next Section Abstract The morphogen retinoic acid promotes the formation of Primitive Endoderm in mouse F9 teratocarcinoma cells as does the stimulation of the Frizzled-1 pathway. We investigated whether the β-catenin/Lef-Tcf-sensitive transcriptional pathway activated by Frizzled-1 plays a role in the retinoic acid-induced pathway to Primitive Endoderm formation. An analysis of Lef-Tcf-sensitive transcription reveals increased transcription at 1 and 4 h post-treatment with retinoic acid. The stimulation of Lef-Tcf-sensitive transcription as well as the formation of Primitive Endoderm was accompanied by the stabilization of β-catenin as observed in activation of the Frizzled-1 pathway. Transient transfection of F9 cells with an expression vector harboring a dominant-negative mutant of Tcf4 resulted in the attenuation of both the increase in Lef-Tcf-sensitive transcription and formation of Primitive Endoderm in response to the morphogen. Clones stably transfected to express the dominant-negative Tcf4 displayed a block in retinoic acid-induced activation of Lef-Tcf-sensitive transcription and Primitive Endoderm formation. These data reveal the obligate role of the β-catenin/Lef-Tcf transcriptional pathway in the action of the morphogen retinoic acid.
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Activation of the β-catenin/Lef-Tcf pathway is obligate for formation of Primitive Endoderm by mouse F9 totipotent teratocarcinoma cells in response to retinoic acid
The Journal of biological chemistry, 2002Co-Authors: Tong Liu, Yi-nan Lee, Craig C. Malbon, Hsien-yu WangAbstract:Next Section Abstract The morphogen retinoic acid promotes the formation of Primitive Endoderm in mouse F9 teratocarcinoma cells as does the stimulation of the Frizzled-1 pathway. We investigated whether the β-catenin/Lef-Tcf-sensitive transcriptional pathway activated by Frizzled-1 plays a role in the retinoic acid-induced pathway to Primitive Endoderm formation. An analysis of Lef-Tcf-sensitive transcription reveals increased transcription at 1 and 4 h post-treatment with retinoic acid. The stimulation of Lef-Tcf-sensitive transcription as well as the formation of Primitive Endoderm was accompanied by the stabilization of β-catenin as observed in activation of the Frizzled-1 pathway. Transient transfection of F9 cells with an expression vector harboring a dominant-negative mutant of Tcf4 resulted in the attenuation of both the increase in Lef-Tcf-sensitive transcription and formation of Primitive Endoderm in response to the morphogen. Clones stably transfected to express the dominant-negative Tcf4 displayed a block in retinoic acid-induced activation of Lef-Tcf-sensitive transcription and Primitive Endoderm formation. These data reveal the obligate role of the β-catenin/Lef-Tcf transcriptional pathway in the action of the morphogen retinoic acid.
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Expression of Gα13 (Q226L) Induces P19 Stem Cells to Primitive Endoderm via MEKK1, 2, or 4
The Journal of biological chemistry, 2001Co-Authors: Hsien-yu Wang, Jyotshnabala Kanungo, Craig C. MalbonAbstract:Galpha13 mediates the ability of the morphogen retinoic acid to promote Primitive Endoderm formation from mouse P19 embryonal carcinoma stem cells, a process that includes the obligate activation of Jun N-terminal kinase. Expression of the constitutively activated (Q226L) GTPase-deficient form of Galpha13 mimics retinoic acid and was used to investigate the signaling upstream of Primitive Endoderm formation. Jun N-terminal kinase 1 activity, MEK1,2, MKK4, and MEKK1 were constitutively activated in clones stably transfected to express Q226L Galpha13. Dominant negative forms of MEKK1 and MEKK4 were expressed stably in the clones harboring Q226L Galpha13. Expression of dominant negative versions of either MEKK1 or MEKK4 effectively blocks both the activation of Jun N-terminal kinase as well as the formation of Primitive Endoderm. Depletion of MEKK1, -2, or -4 by antisense oligodeoxynucleotides suppressed signaling from Q226L Galpha13 to JNK1 and Primitive Endoderm formation. We demonstrate that the signal linkage map from Galpha13 activation to Primitive Endoderm formation in these stem cells requires activation at three levels of the mitogen-activated protein kinase cascade: MEKK1, -2, or -4 for MAP kinase kinase kinase; MKK4 and/or MEK1 for MAP kinase kinase; and JNK1 for MAP kinase.
Hélène Jacquemin-sablon - One of the best experts on this subject based on the ideXlab platform.
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The RNA-Binding Protein Unr Prevents Mouse Embryonic Stem Cells Differentiation Toward the Primitive Endoderm Lineage
STEM CELLS, 2011Co-Authors: Habiba Elatmani, Claire Chazaud, Virginie Dormoy-raclet, Pierre Dubus, François Dautry, Hélène Jacquemin-sablonAbstract:The maintenance of embryonic stem cells (ESCs) pluripotency depends on key transcription factors, chromatin remodeling proteins, and microRNAs. The roles of RNA-binding proteins are however poorly understood. We report that the cytoplasmic RNA-binding protein Unr prevents the differentiation of ESCs into Primitive Endoderm (PrE). We show that unr knockout (unr(-/-) ) ESCs spontaneously differentiate into PrE, and that Unr re-expression in unr(-/-) ESCs reverses this phenotype. Nevertheless, unr(-/-) ESCs retain pluripotency, producing differentiated teratomas, and the differentiated unr(-/-) ESCs coexpress the PrE inducer Gata6 and the pluripotency factors Oct4, Nanog, and Sox2. Interestingly, in the differentiated unr(-/-) ESCs, Nanog and Sox2 exhibit a dual nuclear and cytoplasmic localization. This situation, that has never been reported, likely reflects an early differentiation state toward PrE. Finally, we show that Unr destabilizes Gata6 mRNAs and we propose that the post-transcriptional repression of Gata6 expression by Unr contributes to the stabilization of the ESCs pluripotent state.
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The RNA-binding protein Unr prevents mouse embryonic stem cells differentiation toward the Primitive Endoderm lineage.
Stem cells (Dayton Ohio), 2011Co-Authors: Habiba Elatmani, Claire Chazaud, Virginie Dormoy-raclet, Pierre Dubus, François Dautry, Hélène Jacquemin-sablonAbstract:The maintenance of embryonic stem cells (ESCs) pluripotency depends on key transcription factors, chromatin remodeling proteins, and microRNAs. The roles of RNA-binding proteins are however poorly understood. We report that the cytoplasmic RNA-binding protein Unr prevents the differentiation of ESCs into Primitive Endoderm (PrE). We show that unr knockout (unr−/−) ESCs spontaneously differentiate into PrE, and that Unr re-expression in unr−/− ESCs reverses this phenotype. Nevertheless, unr−/− ESCs retain pluripotency, producing differentiated teratomas, and the differentiated unr−/− ESCs coexpress the PrE inducer Gata6 and the pluripotency factors Oct4, Nanog, and Sox2. Interestingly, in the differentiated unr−/− ESCs, Nanog and Sox2 exhibit a dual nuclear and cytoplasmic localization. This situation, that has never been reported, likely reflects an early differentiation state toward PrE. Finally, we show that Unr destabilizes Gata6 mRNAs and we propose that the post-transcriptional repression of Gata6 expression by Unr contributes to the stabilization of the ESCs pluripotent state. STEM CELLS 2011;29:1504–1516
Annakaterina Hadjantonakis - One of the best experts on this subject based on the ideXlab platform.
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gata6 levels modulate Primitive Endoderm cell fate choice and timing in the mouse blastocyst
Developmental Cell, 2014Co-Authors: Nadine Schrode, Nestor Saiz, Stefano Di Talia, Annakaterina HadjantonakisAbstract:Cells of the inner cell mass (ICM) of the mouse blastocyst differentiate into the pluripotent epiblast or the Primitive Endoderm (PrE), marked by the transcription factors NANOG and GATA6, respectively. To investigate the mechanistic regulation of this process, we applied an unbiased, quantitative, single-cell-resolution image analysis pipeline to analyze embryos lacking or exhibiting reduced levels of GATA6. We find that Gata6 mutants exhibit a complete absence of PrE and demonstrate that GATA6 levels regulate the timing and speed of lineage commitment within the ICM. Furthermore, we show that GATA6 is necessary for PrE specification by FGF signaling and propose a model where interactions between NANOG, GATA6, and the FGF/ERK pathway determine ICM cell fate. This study provides a framework for quantitative analyses of mammalian embryos and establishes GATA6 as a nodal point in the gene regulatory network driving ICM lineage specification.
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PDGF signaling is required for Primitive Endoderm cell survival in the inner cell mass of the mouse blastocyst
STEM CELLS, 2013Co-Authors: Jérôme Artus, Michel Cohen-tannoudji, Minjung Kang, Annakaterina HadjantonakisAbstract:At the end of the preimplantation period, the inner cell mass (ICM) of the mouse blastocyst is composed of two distinct cell lineages, the pluripotent epiblast (EPI) and the Primitive Endoderm (PrE). The current model for their formation involves initial co-expression of lineage-specific markers followed by mutual-exclusive expression resulting in a salt-and-pepper distribution of lineage precursors within the ICM. Subsequent to lineage commitment, cell rearrangements and selective apoptosis are thought to be key processes driving and refining the emergence of two spatially distinct compartments. Here, we have addressed a role for Platelet Derived Growth Factor (PDGF) signaling in the regulation of programmed cell death during early mouse embryonic development. By combining genetic and pharmacological approaches, we demonstrate that embryos lacking PDGF activity exhibited caspase-dependent selective apoptosis of PrE cells. Modulating PDGF activity did not affect lineage commitment or cell sorting, suggesting that PDGF is involved in the fine-tuning of patterning information. Our results also indicate that PDGF and fibroblast growth factor (FGF) tyrosine kinase receptors exert distinct and non-overlapping functions in PrE formation. Taken together, these data uncover an early role of PDGF signaling in PrE cell survival at the time when PrE and EPI cells are segregated.
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FGF4 is required for lineage restriction and salt-and-pepper distribution of Primitive Endoderm factors but not their initial expression in the mouse
Development (Cambridge England), 2012Co-Authors: Minjung Kang, Anna Piliszek, Jérôme Artus, Annakaterina HadjantonakisAbstract:The emergence of pluripotent epiblast (EPI) and Primitive Endoderm (PrE) lineages within the inner cell mass (ICM) of the mouse blastocyst involves initial co-expression of lineage-associated markers followed by mutual exclusion and salt-and-pepper distribution of lineage-biased cells. Precisely how EPI and PrE cell fate commitment occurs is not entirely clear; however, previous studies in mice have implicated FGF/ERK signaling in this process. Here, we investigated the phenotype resulting from zygotic and maternal/zygotic inactivation of Fgf4. Fgf4 heterozygous blastocysts exhibited increased numbers of NANOG-positive EPI cells and reduced numbers of GATA6-positive PrE cells, suggesting that FGF signaling is tightly regulated to ensure specification of the appropriate numbers of cells for each lineage. Although the size of the ICM was unaffected in Fgf4 null mutant embryos, it entirely lacked a PrE layer and exclusively comprised NANOG-expressing cells at the time of implantation. An initial period of widespread EPI and PrE marker co-expression was however established even in the absence of FGF4. Thus, Fgf4 mutant embryos initiated the PrE program but exhibited defects in its restriction phase, when lineage bias is acquired. Consistent with this, XEN cells could be derived from Fgf4 mutant embryos in which PrE had been restored and these cells appeared indistinguishable from wild-type cells. Sustained exogenous FGF failed to rescue the mutant phenotype. Instead, depending on concentration, we noted no effect or conversion of all ICM cells to GATA6-positive PrE. We propose that heterogeneities in the availability of FGF produce the salt-and-pepper distribution of lineage-biased cells.
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Differential plasticity of epiblast and Primitive Endoderm precursors within the ICM of the early mouse embryo
Development (Cambridge England), 2011Co-Authors: Joanna B. Grabarek, Anna Piliszek, Stephen Frankenberg, Nestor Saiz, Annakaterina Hadjantonakis, Krystyna Zyzyńska, Jennifer Nichols, Berenika PlusaAbstract:Cell differentiation during pre-implantation mammalian development involves the formation of two extra-embryonic lineages: trophoblast and Primitive Endoderm (PrE). A subset of cells within the inner cell mass (ICM) of the blastocyst does not respond to differentiation signals and forms the pluripotent epiblast, which gives rise to all of the tissues in the adult body. How this group of cells is set aside remains unknown. Recent studies documented distinct sequential phases of marker expression during the segregation of epiblast and PrE within the ICM. However, the connection between marker expression and lineage commitment remains unclear. Using a fluorescent reporter for PrE, we investigated the plasticity of epiblast and PrE precursors. Our observations reveal that loss of plasticity does not coincide directly with lineage restriction of epiblast and PrE markers, but rather with exclusion of the pluripotency marker Oct4 from the PrE. We note that individual ICM cells can contribute to all three lineages of the blastocyst until peri-implantation. However, epiblast precursors exhibit less plasticity than precursors of PrE, probably owing to differences in responsiveness to extracellular signalling. We therefore propose that the early embryo environment restricts the fate choice of epiblast but not PrE precursors, thus ensuring the formation and preservation of the pluripotent foetal lineage.
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BMP4 signaling directs Primitive Endoderm-derived XEN cells to an extraembryonic visceral Endoderm identity.
Developmental biology, 2011Co-Authors: Jérôme Artus, Anna Piliszek, Panagiotis Douvaras, Joan Isern, Margaret H. Baron, Annakaterina HadjantonakisAbstract:The visceral Endoderm (VE) is an epithelial tissue in the early postimplantation mouse embryo that encapsulates the pluripotent epiblast distally and the extraembryonic ectoderm proximally. In addition to facilitating nutrient exchange before the establishment of a circulation, the VE is critical for patterning the epiblast. Since VE is derived from the Primitive Endoderm (PrE) of the blastocyst, and PrE-derived eXtraembryonic Endoderm (XEN) cells can be propagated in vitro, XEN cells should provide an important tool for identifying factors that direct VE differentiation. In this study, we demonstrated that BMP4 signaling induces the formation of a polarized epithelium in XEN cells. This morphological transition was reversible, and was associated with the acquisition of a molecular signature comparable to extraembryonic (ex) VE. Resembling exVE which will form the Endoderm of the visceral yolk sac, BMP4-treated XEN cells regulated hematopoiesis by stimulating the expansion of Primitive erythroid progenitors. We also observed that LIF exerted an antagonistic effect on BMP4-induced XEN cell differentiation, thereby impacting the extrinsic conditions used for the isolation and maintenance of XEN cells in an undifferentiated state. Taken together, our data suggest that XEN cells can be differentiated towards an exVE identity upon BMP4 stimulation and therefore represent a valuable tool for investigating PrE lineage differentiation.