RESUMO
The placenta establishes a maternal-fetal exchange interface to transport nutrients and gases between the mother and the fetus. Establishment of this exchange interface relies on the development of multinucleated syncytiotrophoblasts (SynT) from trophoblast progenitors, and defect in SynT development often leads to pregnancy failure and impaired embryonic development. Here, we show that mouse embryos with conditional deletion of transcription factors GATA2 and GATA3 in labyrinth trophoblast progenitors (LaTPs) have underdeveloped placenta and die by ~embryonic day 9.5. Single-cell RNA sequencing analysis revealed excessive accumulation of multipotent LaTPs upon conditional deletion of GATA factors. The GATA factor-deleted multipotent progenitors were unable to differentiate into matured SynTs. We also show that the GATA factor-mediated priming of trophoblast progenitors for SynT differentiation is a conserved event during human placentation. Loss of either GATA2 or GATA3 in cytotrophoblast-derived human trophoblast stem cells (human TSCs) drastically inhibits SynT differentiation potential. Identification of GATA2 and GATA3 target genes along with comparative bioinformatics analyses revealed that GATA factors directly regulate hundreds of common genes in human TSCs, including genes that are essential for SynT development and implicated in preeclampsia and fetal growth retardation. Thus, our study uncovers a conserved molecular mechanism, in which coordinated function of GATA2 and GATA3 promotes trophoblast progenitor-to-SynT commitment, ensuring establishment of the maternal-fetal exchange interface.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Troca Materno-Fetal , Gravidez , Feminino , Humanos , Animais , Camundongos , Placenta , Trofoblastos , Diferenciação Celular/fisiologia , Desenvolvimento Fetal , Fatores de Transcrição GATARESUMO
Healthy progression of human pregnancy relies on cytotrophoblast (CTB) progenitor self-renewal and its differentiation toward multinucleated syncytiotrophoblasts (STBs) and invasive extravillous trophoblasts (EVTs). However, the underlying molecular mechanisms that fine-tune CTB self-renewal or direct its differentiation toward STBs or EVTs during human placentation are poorly defined. Here, we show that Hippo signaling cofactor WW domain containing transcription regulator 1 (WWTR1) is a master regulator of trophoblast fate choice during human placentation. Using human trophoblast stem cells (human TSCs), primary CTBs, and human placental explants, we demonstrate that WWTR1 promotes self-renewal in human CTBs and is essential for their differentiation to EVTs. In contrast, WWTR1 prevents induction of the STB fate in undifferentiated CTBs. Our single-cell RNA sequencing analyses in first-trimester human placenta, along with mechanistic analyses in human TSCs revealed that WWTR1 fine-tunes trophoblast fate by directly regulating WNT signaling components. Importantly, our analyses of placentae from pathological pregnancies show that extreme preterm births (gestational time ≤28 wk) are often associated with loss of WWTR1 expression in CTBs. In summary, our findings establish the critical importance of WWTR1 at the crossroads of human trophoblast progenitor self-renewal versus differentiation. It plays positive instructive roles in promoting CTB self-renewal and EVT differentiation and safeguards undifferentiated CTBs from attaining the STB fate.
Assuntos
Placenta , Placentação , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Trofoblastos , Diferenciação Celular , Feminino , Via de Sinalização Hippo , Humanos , Recém-Nascido , Placenta/metabolismo , Placentação/fisiologia , Gravidez , Nascimento Prematuro/fisiopatologia , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional/genética , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional/metabolismo , Trofoblastos/citologia , Trofoblastos/metabolismoRESUMO
Early pregnancy loss affects â¼15% of all implantation-confirmed human conceptions. However, evolutionarily conserved molecular mechanisms that regulate self-renewal of trophoblast progenitors and their association with early pregnancy loss are poorly understood. Here, we provide evidence that transcription factor TEAD4 ensures survival of postimplantation mouse and human embryos by controlling self-renewal and stemness of trophoblast progenitors within the placenta primordium. In an early postimplantation mouse embryo, TEAD4 is selectively expressed in trophoblast stem cell-like progenitor cells (TSPCs), and loss of Tead4 in postimplantation mouse TSPCs impairs their self-renewal, leading to embryonic lethality before embryonic day 9.0, a developmental stage equivalent to the first trimester of human gestation. Both TEAD4 and its cofactor, yes-associated protein 1 (YAP1), are specifically expressed in cytotrophoblast (CTB) progenitors of a first-trimester human placenta. We also show that a subset of unexplained recurrent pregnancy losses (idiopathic RPLs) is associated with impaired TEAD4 expression in CTB progenitors. Furthermore, by establishing idiopathic RPL patient-specific human trophoblast stem cells (RPL-TSCs), we show that loss of TEAD4 is associated with defective self-renewal in RPL-TSCs and rescue of TEAD4 expression restores their self-renewal ability. Unbiased genomics studies revealed that TEAD4 directly regulates expression of key cell cycle genes in both mouse and human TSCs and establishes a conserved transcriptional program. Our findings show that TEAD4, an effector of the Hippo signaling pathway, is essential for the establishment of pregnancy in a postimplantation mammalian embryo and indicate that impairment of the Hippo signaling pathway could be a molecular cause for early human pregnancy loss.
Assuntos
Autorrenovação Celular/genética , Proteínas de Ligação a DNA/genética , Desenvolvimento Embrionário/genética , Proteínas Musculares/genética , Fatores de Transcrição/genética , Trofoblastos/citologia , Trofoblastos/metabolismo , Aborto Habitual/etiologia , Aborto Habitual/metabolismo , Aborto Espontâneo/etiologia , Aborto Espontâneo/metabolismo , Animais , Biomarcadores , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Suscetibilidade a Doenças , Implantação do Embrião , Feminino , Imunofluorescência , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Imuno-Histoquímica , Camundongos , Proteínas Musculares/metabolismo , Placenta/metabolismo , Gravidez , Fatores de Transcrição de Domínio TEA , Fatores de Transcrição/metabolismoRESUMO
In utero mammalian development relies on the establishment of the maternal-fetal exchange interface, which ensures transportation of nutrients and gases between the mother and the fetus. This exchange interface is established via development of multinucleated syncytiotrophoblast cells (SynTs) during placentation. In mice, SynTs develop via differentiation of the trophoblast stem cell-like progenitor cells (TSPCs) of the placenta primordium, and in humans, SynTs are developed via differentiation of villous cytotrophoblast (CTB) progenitors. Despite the critical need in pregnancy progression, conserved signaling mechanisms that ensure SynT development are poorly understood. Herein, we show that atypical protein kinase C iota (PKCλ/ι) plays an essential role in establishing the SynT differentiation program in trophoblast progenitors. Loss of PKCλ/ι in the mouse TSPCs abrogates SynT development, leading to embryonic death at approximately embryonic day 9.0 (E9.0). We also show that PKCλ/ι-mediated priming of trophoblast progenitors for SynT differentiation is a conserved event during human placentation. PKCλ/ι is selectively expressed in the first-trimester CTBs of a developing human placenta. Furthermore, loss of PKCλ/ι in CTB-derived human trophoblast stem cells (human TSCs) impairs their SynT differentiation potential both in vitro and after transplantation in immunocompromised mice. Our mechanistic analyses indicate that PKCλ/ι signaling maintains expression of GCM1, GATA2, and PPARγ, which are key transcription factors to instigate SynT differentiation programs in both mouse and human trophoblast progenitors. Our study uncovers a conserved molecular mechanism, in which PKCλ/ι signaling regulates establishment of the maternal-fetal exchange surface by promoting trophoblast progenitor-to-SynT transition during placentation.
Assuntos
Diferenciação Celular/fisiologia , Isoenzimas/metabolismo , Troca Materno-Fetal/fisiologia , Placenta/metabolismo , Proteína Quinase C/metabolismo , Trofoblastos/fisiologia , Animais , Proteínas de Ligação a DNA/metabolismo , Feminino , Fator de Transcrição GATA2/metabolismo , Humanos , Isoenzimas/genética , Masculino , Camundongos , Camundongos Knockout , Modelos Animais , PPAR gama/metabolismo , Placenta/citologia , Placentação/fisiologia , Gravidez , Proteína Quinase C/genética , Transdução de Sinais , Células-Tronco/citologia , Fatores de Transcrição/metabolismo , Trofoblastos/citologiaRESUMO
Early mammalian development is crucially dependent on the establishment of oxidative energy metabolism within the trophectoderm (TE) lineage. Unlike the inner cell mass, TE cells enhance ATP production via mitochondrial oxidative phosphorylation (OXPHOS) and this metabolic preference is essential for blastocyst maturation. However, molecular mechanisms that regulate establishment of oxidative energy metabolism in TE cells are incompletely understood. Here, we show that conserved transcription factor TEAD4, which is essential for pre-implantation mammalian development, regulates this process by promoting mitochondrial transcription. In developing mouse TE and TE-derived trophoblast stem cells (TSCs), TEAD4 localizes to mitochondria, binds to mitochondrial DNA (mtDNA) and facilitates its transcription by recruiting mitochondrial RNA polymerase (POLRMT). Loss of TEAD4 impairs recruitment of POLRMT, resulting in reduced expression of mtDNA-encoded electron transport chain components, thereby inhibiting oxidative energy metabolism. Our studies identify a novel TEAD4-dependent molecular mechanism that regulates energy metabolism in the TE lineage to ensure mammalian development.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Desenvolvimento Embrionário/genética , Metabolismo Energético , Mamíferos/embriologia , Mamíferos/genética , Mitocôndrias/genética , Proteínas Musculares/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Animais , Blastocisto/citologia , Blastocisto/metabolismo , Blastocisto/ultraestrutura , DNA Mitocondrial/genética , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Ectoderma/citologia , Transporte de Elétrons , Metabolismo Energético/genética , Camundongos , Mitocôndrias/ultraestrutura , Modelos Biológicos , Proteínas Musculares/deficiência , Proteínas Musculares/genética , Oxirredução , Células-Tronco/citologia , Células-Tronco/metabolismo , Fatores de Transcrição de Domínio TEA , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Trofoblastos/citologiaRESUMO
A successful pregnancy is critically dependent upon proper placental development and function. During human placentation, villous cytotrophoblast (CTB) progenitors differentiate to form syncytiotrophoblasts (SynTBs), which provide the exchange surface between the mother and fetus and secrete hormones to ensure proper progression of pregnancy. However, epigenetic mechanisms that regulate SynTB differentiation from CTB progenitors are incompletely understood. Here, we show that lysine-specific demethylase 1 (LSD1; also known as KDM1A), a histone demethylase, is essential to this process. LSD1 is expressed both in CTB progenitors and differentiated SynTBs in first-trimester placental villi; accordingly, expression in SynTBs is maintained throughout gestation. Impairment of LSD1 function in trophoblast progenitors inhibits induction of endogenous retrovirally encoded genes SYNCYTIN1/endogenous retrovirus group W member 1, envelope (ERVW1) and SYNCYTIN2/endogenous retrovirus group FRD member 1, envelope (ERVFRD1), encoding fusogenic proteins critical to human trophoblast syncytialization. Loss of LSD1 also impairs induction of chorionic gonadotropin α (CGA) and chorionic gonadotropin ß (CGB) genes, which encode α and ß subunits of human chorionic gonadotrophin (hCG), a hormone essential to modulate maternal physiology during pregnancy. Mechanistic analyses at the endogenous ERVW1, CGA, and CGB loci revealed a regulatory axis in which LSD1 induces demethylation of repressive histone H3 lysine 9 dimethylation (H3K9Me2) and interacts with transcription factor GATA2 to promote RNA polymerase II (RNA-POL-II) recruitment and activate gene transcription. Our study reveals a novel LSD1-GATA2 axis, which regulates human trophoblast syncytialization.
Assuntos
Diferenciação Celular/genética , Fator de Transcrição GATA2/genética , Histona Desmetilases/genética , Trofoblastos/metabolismo , Vilosidades Coriônicas/crescimento & desenvolvimento , Vilosidades Coriônicas/metabolismo , Epigênese Genética/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Produtos do Gene env/genética , Humanos , Relações Mãe-Filho , Placentação/genética , Gravidez , Proteínas da Gravidez/genética , RNA Polimerase II/genética , Transdução de Sinais/genéticaRESUMO
GATA transcription factors are implicated in establishing cell fate during mammalian development. In early mammalian embryos, GATA3 is selectively expressed in the extraembryonic trophoblast lineage and regulates gene expression to promote trophoblast fate. However, trophoblast-specific GATA3 function is dispensable for early mammalian development. Here, using dual conditional knockout mice, we show that genetic redundancy of Gata3 with paralog Gata2 in trophoblast progenitors ensures the successful progression of both pre- and postimplantation mammalian development. Stage-specific gene deletion in trophoblasts reveals that loss of both GATA genes, but not either alone, leads to embryonic lethality prior to the onset of their expression within the embryo proper. Using ChIP-seq and RNA-seq analyses, we define the global targets of GATA2/GATA3 and show that they directly regulate a large number of common genes to orchestrate stem versus differentiated trophoblast fate. In trophoblast progenitors, GATA factors directly regulate BMP4, Nodal and Wnt signaling components that promote embryonic-extraembryonic signaling cross-talk, which is essential for the development of the embryo proper. Our study provides genetic evidence that impairment of trophoblast-specific GATA2/GATA3 function could lead to early pregnancy failure.
Assuntos
Fator de Transcrição GATA2/fisiologia , Fator de Transcrição GATA3/fisiologia , Placenta/fisiologia , Células-Tronco/citologia , Trofoblastos/citologia , Animais , Diferenciação Celular , Linhagem da Célula , Implantação do Embrião , Desenvolvimento Embrionário , Feminino , Deleção de Genes , Humanos , Camundongos , Camundongos Knockout , Gravidez , Prenhez , Análise de Sequência de RNARESUMO
Cell fate decisions are fundamental to the development of multicellular organisms. In mammals the first cell fate decision involves segregation of the pluripotent inner cell mass and the trophectoderm, a process regulated by cell polarity proteins, HIPPO signaling and lineage-specific transcription factors such as CDX2. However, the regulatory mechanisms that operate upstream to specify the trophectoderm lineage have not been established. Here we report that transcription factor AP-2γ (TFAP2C) functions as a novel upstream regulator of Cdx2 expression and position-dependent HIPPO signaling in mice. Loss- and gain-of-function studies and promoter analysis revealed that TFAP2C binding to an intronic enhancer is required for activation of Cdx2 expression during early development. During the 8-cell to morula transition TFAP2C potentiates cell polarity to suppress HIPPO signaling in the outside blastomeres. TFAP2C depletion triggered downregulation of PARD6B, loss of apical cell polarity, disorganization of F-actin, and activation of HIPPO signaling in the outside blastomeres. Rescue experiments using Pard6b mRNA restored cell polarity but only partially corrected position-dependent HIPPO signaling, suggesting that TFAP2C negatively regulates HIPPO signaling via multiple pathways. Several genes involved in regulation of the actin cytoskeleton (including Rock1, Rock2) were downregulated in TFAP2C-depleted embryos. Inhibition of ROCK1 and ROCK2 activity during the 8-cell to morula transition phenocopied TFAP2C knockdown, triggering a loss of position-dependent HIPPO signaling and decrease in Cdx2 expression. Altogether, these results demonstrate that TFAP2C facilitates trophectoderm lineage specification by functioning as a key regulator of Cdx2 transcription, cell polarity and position-dependent HIPPO signaling.
Assuntos
Diferenciação Celular/fisiologia , Linhagem da Célula/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Homeodomínio/metabolismo , Transdução de Sinais/fisiologia , Fator de Transcrição AP-2/metabolismo , Fatores de Transcrição/metabolismo , Trofoblastos/fisiologia , Amidas/farmacologia , Análise de Variância , Animais , Fator de Transcrição CDX2 , Polaridade Celular/fisiologia , Imunoprecipitação da Cromatina , Regulação da Expressão Gênica no Desenvolvimento/genética , Via de Sinalização Hippo , Luciferases , Camundongos , Microscopia de Fluorescência , Proteínas Serina-Treonina Quinases/metabolismo , Piridinas/farmacologia , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/genética , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/metabolismoRESUMO
Umbilical cord blood (UCB) engraftment is in part limited by graft cell dose, generally one log less than that of bone marrow (BM)/peripheral blood (PB) cell grafts. Strategies toward increasing hematopoietic stem/progenitor cell (HSPC) homing to BM have been assessed to improve UCB engraftment. Despite recent progress, a complete understanding of how HSPC homing and engraftment are regulated is still elusive. We provide evidence that blocking erythropoietin (EPO)-EPO receptor (R) signaling promotes homing to BM and early engraftment of UCB CD34+ cells. A significant population of UCB CD34+ HSPC expresses cell surface EPOR. Exposure of UCB CD34+ HSPC to EPO inhibits their migration and enhances erythroid differentiation. This migratory inhibitory effect was reversed by depleting EPOR expression on HSPC. Moreover, systemic reduction in EPO levels by hyperbaric oxygen (HBO) used in a preclinical mouse model and in a pilot clinical trial promoted homing of transplanted UCB CD34+ HSPC to BM. Such a systemic reduction of EPO in the host enhanced myeloid differentiation and improved BM homing of UCB CD34+ cells, an effect that was overcome with exogenous EPO administration. Of clinical relevance, HBO therapy before human UCB transplantation was well-tolerated and resulted in transient reduction in EPO with encouraging engraftment rates and kinetics. Our studies indicate that systemic reduction of EPO levels in the host or blocking EPO-EPOR signaling may be an effective strategy to improve BM homing and engraftment after allogeneic UCB transplantation. This clinical trial was registered at www.ClinicalTrials.gov (#NCT02099266).
Assuntos
Transplante de Células-Tronco de Sangue do Cordão Umbilical , Eritropoetina/metabolismo , ADP-Ribosil Ciclase 1/metabolismo , Adolescente , Adulto , Idoso , Animais , Antígenos CD34/metabolismo , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Quimiocina CXCL12/farmacologia , Quimerismo , Estudos de Coortes , Transplante de Células-Tronco de Sangue do Cordão Umbilical/efeitos adversos , Feminino , Doença Enxerto-Hospedeiro/etiologia , Humanos , Oxigenoterapia Hiperbárica , Masculino , Camundongos , Pessoa de Meia-Idade , Células Mieloides/citologia , Células Mieloides/efeitos dos fármacos , Células Mieloides/metabolismo , Receptores da Eritropoetina/metabolismo , Análise de Sobrevida , Condicionamento Pré-Transplante , Resultado do Tratamento , Adulto JovemRESUMO
Pluripotent stem cells (PSCs) contain functionally immature mitochondria and rely upon high rates of glycolysis for their energy requirements. Thus, altered mitochondrial function and promotion of aerobic glycolysis are key to maintain and induce pluripotency. However, signaling mechanisms that regulate mitochondrial function and reprogram metabolic preferences in self-renewing versus differentiated PSC populations are poorly understood. Here, using murine embryonic stem cells (ESCs) as a model system, we demonstrate that atypical protein kinase C isoform, PKC lambda/iota (PKCλ/ι), is a key regulator of mitochondrial function in ESCs. Depletion of PKCλ/ι in ESCs maintains their pluripotent state as evident from germline offsprings. Interestingly, loss of PKCλ/ι in ESCs leads to impairment in mitochondrial maturation, organization, and a metabolic shift toward glycolysis under differentiating condition. Our mechanistic analyses indicate that a PKCλ/ι-hypoxia-inducible factor 1α-PGC1α axis regulates mitochondrial respiration and balances pluripotency in ESCs. We propose that PKCλ/ι could be a crucial regulator of mitochondrial function and energy metabolism in stem cells and other cellular contexts.
Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Embrionárias/metabolismo , Metabolismo Energético/fisiologia , Isoenzimas/metabolismo , Mitocôndrias/metabolismo , Células-Tronco Pluripotentes/metabolismo , Proteína Quinase C/metabolismo , Animais , Glicólise/fisiologia , Humanos , Camundongos , Transdução de Sinais/fisiologiaRESUMO
In the preimplantation mouse embryo, TEAD4 is critical to establishing the trophectoderm (TE)-specific transcriptional program and segregating TE from the inner cell mass (ICM). However, TEAD4 is expressed in the TE and the ICM. Thus, differential function of TEAD4 rather than expression itself regulates specification of the first two cell lineages. We used ChIP sequencing to define genomewide TEAD4 target genes and asked how transcription of TEAD4 target genes is specifically maintained in the TE. Our analyses revealed an evolutionarily conserved mechanism, in which lack of nuclear localization of TEAD4 impairs the TE-specific transcriptional program in inner blastomeres, thereby allowing their maturation toward the ICM lineage. Restoration of TEAD4 nuclear localization maintains the TE-specific transcriptional program in the inner blastomeres and prevents segregation of the TE and ICM lineages and blastocyst formation. We propose that altered subcellular localization of TEAD4 in blastomeres dictates first mammalian cell fate specification.
Assuntos
Linhagem da Célula , Proteínas de Ligação a DNA/metabolismo , Proteínas Musculares/metabolismo , Fatores de Transcrição/metabolismo , Animais , Blastocisto/citologia , Blastocisto/metabolismo , Massa Celular Interna do Blastocisto/citologia , Massa Celular Interna do Blastocisto/metabolismo , Blastômeros/citologia , Blastômeros/metabolismo , Western Blotting , Fator de Transcrição CDX2 , Bovinos , Núcleo Celular/metabolismo , Células Cultivadas , Proteínas de Ligação a DNA/genética , Células-Tronco Embrionárias/metabolismo , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Macaca mulatta , Camundongos , Camundongos Transgênicos , Proteínas Musculares/genética , Interferência de RNA , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição de Domínio TEA , Fatores de Transcrição/genéticaRESUMO
Embryonic stem cell (ESC) pluripotency is orchestrated by distinct signaling pathways that are often targeted to maintain ESC self-renewal or their differentiation to other lineages. We showed earlier that inhibition of PKC signaling maintains pluripotency in mouse ESCs. Therefore, in this study, we investigated the importance of protein kinase C signaling in the context of rat ESC (rESC) pluripotency. Here we show that inhibition of PKC signaling is an efficient strategy to establish and maintain pluripotent rESCs and to facilitate reprogramming of rat embryonic fibroblasts to rat induced pluripotent stem cells. The complete developmental potential of rESCs was confirmed with viable chimeras and germ line transmission. Our molecular analyses indicated that inhibition of a PKCζ-NF-κB-microRNA-21/microRNA-29 regulatory axis contributes to the maintenance of rESC self-renewal. In addition, PKC inhibition maintains ESC-specific epigenetic modifications at the chromatin domains of pluripotency genes and, thereby, maintains their expression. Our results indicate a conserved function of PKC signaling in balancing self-renewal versus differentiation of both mouse and rat ESCs and indicate that targeting PKC signaling might be an efficient strategy to establish ESCs from other mammalian species.
Assuntos
Células-Tronco Embrionárias/enzimologia , Células-Tronco Pluripotentes/enzimologia , Proteína Quinase C-épsilon/metabolismo , Transdução de Sinais/fisiologia , Animais , Células-Tronco Embrionárias/citologia , Indóis/farmacologia , Maleimidas/farmacologia , MicroRNAs/metabolismo , NF-kappa B/metabolismo , Células-Tronco Pluripotentes/citologia , Proteína Quinase C-épsilon/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Ratos , Transdução de Sinais/efeitos dos fármacosRESUMO
Adenosine A1 receptors (A1Rs) are implied in the modulation of neuroinflammation. Activation of cerebral A1Rs acts as a brake on the microglial response after traumatic brain injury and has neuroprotective properties in animal models of Parkinson's disease and multiple sclerosis. Neuroinflammatory processes in turn may affect the expression of A1Rs, but the available data is limited and inconsistent. Here, we applied an animal model of encephalitis to assess how neuroinflammation affects the expression of A1Rs. Two groups of animals were studied: Infected rats (n=7) were intranasally inoculated with herpes simplex virus-1 (HSV-1, 1 × 10(7) plaque forming units), sham-infected rats (n=6) received only phosphate-buffered saline. Six or seven days later, microPET scans (60 min with arterial blood sampling) were made using the tracer 8-dicyclopropyl-1-(11)C-methyl-3-propyl-xanthine ((11)C-MPDX). Tracer clearance from plasma and partition coefficient (K1/k2 estimated from a 2-tissue compartment model fit) were not significantly altered after virus infection. PET tracer distribution volume calculated from a Logan plot was significantly increased in the hippocampus (+37%) and medulla (+27%) of virus infected rats. Tracer binding potential (k3/k4 estimated from the model fit) was significantly increased in the cerebellum (+87%) and the medulla (+148%) which may indicate increased A1R expression. This was confirmed by immunohistochemical analysis showing a strong increase of A1R immunoreactivity in the cerebellum of HSV-1-infected rats. Both the quantitative PET data and immunohistochemical analysis indicate that A1Rs are upregulated in brain areas where active virus is present.
Assuntos
Encéfalo/metabolismo , Encefalite Viral/metabolismo , Infecções por HIV/metabolismo , Fatores Imunológicos/metabolismo , Tomografia por Emissão de Pósitrons/veterinária , Receptor A1 de Adenosina/metabolismo , Xantinas/farmacocinética , Animais , Animais não Endogâmicos , Encefalite Viral/diagnóstico por imagem , Infecções por HIV/diagnóstico por imagem , Taxa de Depuração Metabólica , Tomografia por Emissão de Pósitrons/métodos , Compostos Radiofarmacêuticos/farmacocinética , Ratos , Ratos Wistar , Distribuição Tecidual , Regulação para CimaRESUMO
Mammalian reproduction is critically dependent on the trophoblast cell lineage, which assures proper establishment of maternal-fetal interactions during pregnancy. Specification of trophoblast cell lineage begins with the development of the trophectoderm (TE) in preimplantation embryos. Subsequently, other trophoblast cell types arise with the progression of pregnancy. Studies with transgenic animal models as well as trophoblast stem/progenitor cells have implicated distinct transcriptional and epigenetic regulators in trophoblast lineage development. This review focuses on our current understanding of transcriptional and epigenetic mechanisms regulating specification, determination, maintenance and differentiation of trophoblast cells.
Assuntos
Diferenciação Celular/fisiologia , Linhagem da Célula/fisiologia , Placentação/fisiologia , Fatores de Transcrição/fisiologia , Trofoblastos/citologia , Trofoblastos/fisiologia , Animais , Animais Geneticamente Modificados , Proliferação de Células/fisiologia , Metilação de DNA , Desenvolvimento Embrionário/fisiologia , Epigênese Genética/fisiologia , Feminino , Humanos , Camundongos , Modelos Animais , Gravidez , Células-Tronco/citologia , Células-Tronco/fisiologia , Transcrição Gênica/fisiologiaRESUMO
The first cell-fate decision in mammalian preimplantation embryos is the segregation of the inner cell mass (ICM) and trophectoderm (TE) cell lineages. The ICM develops into the embryo proper, whereas the TE ensures embryo implantation and is the source of the extra-embryonic trophoblast cell lineages, which contribute to the functional components of the placenta. The development of a totipotent zygote into a multi-lineage blastocyst is associated with the generation of distinct transcriptional programs. Several key transcription factors participate in the ICM and TE-specific transcriptional networks, and recent studies indicate that post-translational histone modifications as well as ATP-dependent chromatin remodeling complexes converge with these transcriptional networks to regulate ICM and TE lineage specification. This review will discuss our current understanding and future perspectives related to transcriptional and epigenetic regulatory mechanisms that are implicated in the initial mammalian lineage commitment steps, with a focus on events in mice.
Assuntos
Blastocisto , Linhagem da Célula , Montagem e Desmontagem da Cromatina , Epigênese Genética , Animais , Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento , Histonas , CamundongosRESUMO
Methyltransferase-like 3 (METTL3), the catalytic enzyme of methyltransferase complex for m6A methylation of RNA, is essential for mammalian development. However, the importance of METTL3 in human placentation remains largely unexplored. Here, we show that a fine balance of METTL3 function in trophoblast cells is essential for successful human placentation. Both loss-of and gain-in METTL3 functions are associated with adverse human pregnancies. A subset of recurrent pregnancy losses and preterm pregnancies are often associated with loss of METTL3 expression in trophoblast progenitors. In contrast, METTL3 is induced in pregnancies associated with fetal growth restriction (FGR). Our loss of function analyses showed that METTL3 is essential for the maintenance of human TSC self-renewal and their differentiation to extravillous trophoblast cells (EVTs). In contrast, loss of METTL3 in human TSCs promotes syncytiotrophoblast (STB) development. Global analyses of RNA m6A modification and METTL3-RNA interaction in human TSCs showed that METTL3 regulates m6A modifications on the mRNA molecules of critical trophoblast regulators, including GATA2, GATA3, TEAD1, TEAD4, WWTR1, YAP1, TFAP2C and ASCL2, and loss of METTL3 leads to depletion of mRNA molecules of these critical regulators. Importantly, conditional deletion of Mettl3 in trophoblast progenitors of an early post-implantation mouse embryo also leads to arrested self-renewal. Hence, our findings indicate that METLL3 is a conserved epitranscriptomic governor in trophoblast progenitors and ensures successful placentation by regulating their self-renewal and dictating their differentiation fate.
RESUMO
During embryonic development the placental vasculature acts as a major hematopoietic niche, where endothelial to hematopoietic transition ensures emergence of hematopoietic stem cells (HSCs). However, the molecular mechanisms that regulate the placental hematoendothelial niche are poorly understood. Using a parietal trophoblast giant cell (TGC)-specific knockout mouse model and single-cell RNA-sequencing, we show that the paracrine factors secreted by the TGCs are critical in the development of this niche. Disruptions in the TGC-specific paracrine signaling leads to the loss of HSC population and the concomitant expansion of a KDR+/DLL4+/PROM1+ hematoendothelial cell-population in the placenta. Combining single-cell transcriptomics and receptor-ligand pair analyses, we also define the parietal TGC-dependent paracrine signaling network and identify Integrin signaling as a fundamental regulator of this process. Our study elucidates novel mechanisms by which non-autonomous signaling from the primary parietal TGCs maintain the delicate placental hematopoietic-angiogenic balance and ensures embryonic and extraembryonic development.
RESUMO
The retina is highly metabolically active, relying on glucose uptake and aerobic glycolysis. Situated in close contact to photoreceptors, a key function of cells in the retinal pigment epithelium (RPE) is phagocytosis of damaged photoreceptor outer segments (POS). Here we identify RPE as a local source of insulin in the eye that is stimulated by POS phagocytosis. We show that Ins2 messenger RNA and insulin protein are produced by RPE cells and that this production correlates with RPE phagocytosis of POS. Genetic deletion of phagocytic receptors ('loss of function') reduces Ins2, whereas increasing the levels of the phagocytic receptor MerTK ('gain of function') increases Ins2 production in male mice. Contrary to pancreas-derived systemic insulin, RPE-derived local insulin is stimulated during starvation, which also increases RPE phagocytosis. Global or RPE-specific Ins2 gene deletion decreases retinal glucose uptake in starved male mice, dysregulates retinal physiology, causes defects in phototransduction and exacerbates photoreceptor loss in a mouse model of retinitis pigmentosa. Collectively, these data identify RPE cells as a phagocytosis-induced local source of insulin in the retina, with the potential to influence retinal physiology and disease.
Assuntos
Insulina , Receptores Proteína Tirosina Quinases , Masculino , Camundongos , Animais , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Insulina/metabolismo , Retina/metabolismo , Fagocitose/fisiologia , Glucose/metabolismoRESUMO
The intricate molecular mechanisms that regulate ESC pluripotency are incompletely understood. Prior research indicated that activation of the Janus kinase-signal transducer and activator of transcription (STAT3) pathway or inhibition of extracellular signal-regulated kinase/glycogen synthase kinase 3 (ERK/GSK3) signaling maintains mouse ESC (mESC) pluripotency. Here, we demonstrate that inhibition of protein kinase C (PKC) isoforms maintains mESC pluripotency without the activation of STAT3 or inhibition of ERK/GSK3 signaling pathways. Our analyses revealed that the atypical PKC isoform, PKCζ plays an important role in inducing lineage commitment in mESCs through a PKCζ-nuclear factor kappa-light-chain-enhancer of activated B cells signaling axis. Furthermore, inhibition of PKC isoforms permits derivation of germline-competent ESCs from mouse blastocysts and also facilitates reprogramming of mouse embryonic fibroblasts toward induced pluripotent stem cells. Our results indicate that PKC signaling is critical to balancing ESC self-renewal and lineage commitment.
Assuntos
Linhagem da Célula , Células-Tronco Embrionárias/metabolismo , Células-Tronco Pluripotentes/metabolismo , Proteína Quinase C/metabolismo , Transdução de Sinais , Animais , Diferenciação Celular/fisiologia , Reprogramação Celular , Células-Tronco Embrionárias/citologia , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Fibroblastos/citologia , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Células-Tronco Pluripotentes Induzidas , Camundongos , NF-kappa B/antagonistas & inibidores , Células-Tronco Pluripotentes/citologia , Proteína Quinase C/antagonistas & inibidores , Interferência de RNA , Fator de Transcrição STAT3/metabolismoRESUMO
Angiogenesis is critically dependent on endothelial cell-specific transcriptional mechanisms. However, the molecular processes that regulate chromatin domains and thereby dictate transcription of key endothelial genes are poorly understood. Here, we report that, in endothelial cells, angiogenic signal-mediated transcriptional induction of Vegfr1 (vascular endothelial growth factor receptor 1) is dependent on the histone chaperone, HIRA (histone cell cycle regulation-defective homolog A). Our molecular analyses revealed that, in response to angiogenic signals, HIRA is induced in endothelial cells and mediates incorporation of lysine 56 acetylated histone H3.3 (H3acK56) at the chromatin domain of Vegfr1. HIRA-mediated incorporation of H3acK56 is a general mechanism associated with transcriptional induction of several angiogenic genes in endothelial cells. Depletion of HIRA inhibits H3acK56 incorporation and transcriptional induction of Vegfr1 and other angiogenic genes. Our functional analyses revealed that depletion of HIRA abrogates endothelial network formation on Matrigel and inhibits angiogenesis in an in vivo Matrigel plug assay. Furthermore, analysis in a laser-induced choroidal neovascularization model showed that depletion of HIRA significantly inhibits neovascularization. Our results for the first time decipher a histone chaperone (HIRA)-dependent molecular mechanism in endothelial gene regulation and indicate that histone chaperones could be new targets for angiogenesis therapy.