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1.
PLoS Genet ; 8(3): e1002609, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22479204

RESUMO

Insulin-like growth factor I receptor (Igf1r) signaling controls proliferation, differentiation, growth, and cell survival in many tissues; and its deregulated activity is involved in tumorigenesis. Although important during fetal growth and postnatal life, a function for the Igf pathway during preimplantation development has not been described. We show that abrogating Igf1r signaling with specific inhibitors blocks trophectoderm formation and compromises embryo survival during murine blastocyst formation. In normal embryos total Igf1r is present throughout the membrane, whereas the activated form is found exclusively at cell contact sites, colocalizing with E-cadherin. Using genetic domain switching, we show a requirement for E-cadherin to maintain proper activation of Igf1r. Embryos expressing exclusively a cadherin chimera with N-cadherin extracellular and E-cadherin intracellular domains (NcEc) fail to form a trophectoderm and cells die by apoptosis. In contrast, homozygous mutant embryos expressing a reverse-structured chimera (EcNc) show trophectoderm survival and blastocoel cavitation, indicating a crucial and non-substitutable role of the E-cadherin ectodomain for these processes. Strikingly, blastocyst formation can be rescued in homozygous NcEc embryos by restoring Igf1r signaling, which enhances cell survival. Hence, perturbation of E-cadherin extracellular integrity, independent of its cell-adhesion function, blocked Igf1r signaling and induced cell death in the trophectoderm. Our results reveal an important and yet undiscovered function of Igf1r during preimplantation development mediated by a unique physical interaction between Igf1r and E-cadherin indispensable for proper receptor activation and anti-apoptotic signaling. We provide novel insights into how ligand-dependent Igf1r activity is additionally gated to sense developmental potential in utero and into a bifunctional role of adhesion molecules in contact formation and signaling.


Assuntos
Blastocisto , Caderinas , Desenvolvimento Embrionário/genética , Receptor IGF Tipo 1 , Transdução de Sinais/genética , Animais , Apoptose , Blastocisto/citologia , Blastocisto/metabolismo , Caderinas/genética , Caderinas/metabolismo , Adesão Celular/genética , Comunicação Celular/genética , Sobrevivência Celular , Células-Tronco Embrionárias , Regulação da Expressão Gênica no Desenvolvimento , Homozigoto , Ligantes , Camundongos , Mutação , Estrutura Terciária de Proteína/genética , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo
2.
Commun Biol ; 7(1): 31, 2024 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-38182651

RESUMO

The stability of cellular phenotypes in developing organisms depends on error-free transmission of epigenetic and genetic information during mitosis. Methylation of cytosine residues in genomic DNA is a key epigenetic mark that modulates gene expression and prevents genome instability. Here, we report on a genetic test of the relationship between DNA replication and methylation in the context of the developing vertebrate organism instead of cell lines. Our analysis is based on the identification of hypomorphic alleles of dnmt1, encoding the DNA maintenance methylase Dnmt1, and pole1, encoding the catalytic subunit of leading-strand DNA polymerase epsilon holoenzyme (Pole). Homozygous dnmt1 mutants exhibit genome-wide DNA hypomethylation, whereas the pole1 mutation is associated with increased DNA methylation levels. In dnmt1/pole1 double-mutant zebrafish larvae, DNA methylation levels are restored to near normal values, associated with partial rescue of mutant-associated transcriptional changes and phenotypes. Hence, a balancing antagonism between DNA replication and maintenance methylation buffers against replicative errors contributing to the robustness of vertebrate development.


Assuntos
Metilação de DNA , Peixe-Zebra , Animais , Peixe-Zebra/genética , Alelos , DNA , Epigênese Genética
3.
Proc Natl Acad Sci U S A ; 107(38): 16613-8, 2010 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-20823228

RESUMO

The thymus is essential for T-cell development. Here, we focus on the role of the transcription factor Foxn1 in the development and function of thymic epithelial cells (TECs) of the mouse. TECs are of endodermal origin; they initially express Foxn1 and give rise to orthotopic (thoracic) and additional (cervical) thymi. Using Foxn1-directed cytoablation, we show that during embryogenesis, cervical thymi develop a few days after the thoracic lobes, and that bipotent epithelial progenitors of cortical and medullary compartments express Foxn1. We also show that following acute selective near-total ablation during embryogenesis, complete regeneration of TECs does not occur, providing an animal model for human thymic aplasia syndromes. Finally, we address the functional role of Foxn1-negative TECs that arise postnatally in the mouse. Lineage tracing shows that such Foxn1-negative TECs are descendants of Foxn1-positive progenitors; furthermore, Foxn1-directed subacute intoxication of TECs by polyglutamine-containing EGFP proteins indicates that a presumptive Foxn1-independent lineage does not contribute to thymopoietic function of the adult thymus. Our findings therefore support the notion that Foxn1 is the essential transcription factor regulating the differentiation of TECs and that its expression marks the major functional lineage of TECs in embryonic and adult thymic tissue.


Assuntos
Fatores de Transcrição Forkhead/metabolismo , Linfopoese/fisiologia , Timo/embriologia , Animais , Sequência de Bases , Primers do DNA/genética , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Fatores de Transcrição Forkhead/deficiência , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Linfopoese/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Gravidez , Timo/anormalidades , Timo/citologia , Timo/metabolismo
4.
Nat Cell Biol ; 25(9): 1265-1278, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37652981

RESUMO

Despite advances in four-factor (4F)-induced reprogramming (4FR) in vitro and in vivo, how 4FR interconnects with senescence remains largely under investigated. Here, using genetic and chemical approaches to manipulate senescent cells, we show that removal of p16High cells resulted in the 4FR of somatic cells into totipotent-like stem cells. These cells expressed markers of both pluripotency and the two-cell embryonic state, readily formed implantation-competent blastoids and, following morula aggregation, contributed to embryonic and extraembryonic lineages. We identified senescence-dependent regulation of nicotinamide N-methyltransferase as a key mechanism controlling the S-adenosyl-L-methionine levels during 4FR that was required for expression of the two-cell genes and acquisition of an extraembryonic potential. Importantly, a partial 4F epigenetic reprogramming in old mice was able to reverse several markers of liver aging only in conjunction with the depletion of p16High cells. Our results show that the presence of p16High senescent cells limits cell plasticity, whereas their depletion can promote a totipotent-like state and histopathological tissue rejuvenation during 4F reprogramming.


Assuntos
Plasticidade Celular , Reprogramação Celular , Animais , Camundongos , Reprogramação Celular/genética , Envelhecimento/genética , Implantação do Embrião , Epigenômica
5.
Am J Pathol ; 177(2): 840-53, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20558576

RESUMO

Tissue inhibitors of metalloproteinases (TIMPs) are a family of closely related proteins that inhibit matrix metalloproteinases (MMPs). In the central nervous system (CNS), TIMPs 2, 3, and 4 are constitutively expressed at high levels, whereas TIMP1 can be induced by various stimuli. Here, we studied the effects of constitutive expression of TIMP1 in the CNS in transgenic mice. Transgene expression started prenatally and persisted throughout lifetime at high levels. Since MMP activity has been implicated in CNS development, in proper function of the adult CNS, and in inflammatory disorders, we investigated Timp1-induced CNS alterations. Despite sufficient MMP inhibition, high expressor transgenic mice had a normal phenotype. The absence of compensatory up-regulation of MMP genes in the CNS of Timp1 transgenic mice indicates that development, learning, and memory functions do not require the entire MMP arsenal. To elucidate the effects of strong Timp1 expression in CNS inflammation, we induced experimental allergic encephalomyelitis. We observed a Timp1 dose-dependent mitigation of both experimental allergic encephalomyelitis symptoms and histological lesions in the CNS of transgenic mice. All in all, our data demonstrate that (1) long-term CNS expression of TIMP1 with complete suppression of gelatinolytic activity does not interfere with physiological brain function and (2) TIMP1 might constitute a promising candidate for long-term therapeutic treatment of inflammatory CNS diseases such as multiple sclerosis.


Assuntos
Sistema Nervoso Central/metabolismo , Encefalomielite Autoimune Experimental/metabolismo , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Animais , Comportamento Animal/fisiologia , Sistema Nervoso Central/patologia , Feminino , Masculino , Metaloproteinases da Matriz/genética , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Transgênicos , Testes Neuropsicológicos , Gravidez , Inibidor Tecidual de Metaloproteinase-1/genética
7.
Proc Natl Acad Sci U S A ; 105(13): 5177-82, 2008 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-18362329

RESUMO

The giant cytosolic protease tripeptidyl peptidase II (TPPII) has been implicated in the regulation of proliferation and survival of malignant cells, particularly lymphoma cells. To address its functions in normal cellular and systemic physiology we have generated TPPII-deficient mice. TPPII deficiency activates cell type-specific death programs, including proliferative apoptosis in several T lineage subsets and premature cellular senescence in fibroblasts and CD8(+) T cells. This coincides with up-regulation of p53 and dysregulation of NF-kappaB. Prominent degenerative alterations at the organismic level were a decreased lifespan and symptoms characteristic of immunohematopoietic senescence. These symptoms include accelerated thymic involution, lymphopenia, impaired proliferative T cell responses, extramedullary hematopoiesis, and inflammation. Thus, TPPII is important for maintaining normal cellular and systemic physiology, which may be relevant for potential therapeutic applications of TPPII inhibitors.


Assuntos
Envelhecimento/imunologia , Apoptose/imunologia , Serina Endopeptidases/deficiência , Serina Endopeptidases/metabolismo , Aminopeptidases , Animais , Diferenciação Celular/imunologia , Células Cultivadas , Dipeptidil Peptidases e Tripeptidil Peptidases , Fibroblastos , Deleção de Genes , Linfopenia/enzimologia , Linfopenia/genética , Linfopenia/patologia , Camundongos , Camundongos Knockout , NF-kappa B/metabolismo , Fenótipo , Serina Endopeptidases/genética , Linfócitos T/citologia , Linfócitos T/enzimologia , Linfócitos T/imunologia , Timo/citologia , Timo/enzimologia , Timo/imunologia , Proteína Supressora de Tumor p53/metabolismo
8.
Dev Cell ; 3(2): 171-81, 2002 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12194849

RESUMO

Using Cre/loxP, we conditionally inactivated the beta-catenin gene in cells of structures that exhibit important embryonic organizer functions: the visceral endoderm, the node, the notochord, and the definitive endoderm. Mesoderm formation was not affected in the mutant embryos, but the node was missing, patterning of the head and trunk was affected, and no notochord or somites were formed. Surprisingly, deletion of beta-catenin in the definitive endoderm led to the formation of multiple hearts all along the anterior-posterior (A/P) axis of the embryo. Ectopic hearts developed in parallel with the normal heart in regions of ectopic Bmp2 expression. We provide evidence that ablation of beta-catenin in embryonic endoderm changes cell fate from endoderm to precardiac mesoderm, consistent with the existence of bipotential mesendodermal progenitors in mouse embryos.


Assuntos
Diferenciação Celular/genética , Linhagem da Célula/genética , Coristoma/genética , Proteínas do Citoesqueleto/deficiência , Embrião de Mamíferos/anormalidades , Deleção de Genes , Cardiopatias Congênitas/genética , Camundongos Knockout/anormalidades , Transativadores/deficiência , Animais , Padronização Corporal/genética , Células Cultivadas , Quimera/anormalidades , Quimera/genética , Quimera/metabolismo , Coristoma/metabolismo , Coristoma/fisiopatologia , Citocinas , Proteínas do Citoesqueleto/genética , Proteínas de Ligação a DNA/metabolismo , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Endoderma/citologia , Endoderma/metabolismo , Feminino , Fator de Transcrição GATA4 , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Cabeça/anormalidades , Cardiopatias Congênitas/metabolismo , Cardiopatias Congênitas/fisiopatologia , Proteínas de Homeodomínio/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Mesoderma/citologia , Mesoderma/metabolismo , Camundongos , Camundongos Knockout/genética , Camundongos Knockout/metabolismo , Mutação/genética , Proteínas/metabolismo , Transativadores/genética , Fatores de Transcrição/metabolismo , beta Catenina
9.
Dev Cell ; 4(3): 395-406, 2003 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-12636920

RESUMO

The vertebral column derives from somites generated by segmentation of presomitic mesoderm (PSM). Somitogenesis involves a molecular oscillator, the segmentation clock, controlling periodic Notch signaling in the PSM. Here, we establish a novel link between Wnt/beta-catenin signaling and the segmentation clock. Axin2, a negative regulator of the Wnt pathway, is directly controlled by Wnt/beta-catenin and shows oscillating expression in the PSM, even when Notch signaling is impaired, alternating with Lfng expression. Moreover, Wnt3a is required for oscillating Notch signaling activity in the PSM. We propose that the segmentation clock is established by Wnt/beta-catenin signaling via a negative-feedback mechanism and that Wnt3a controls the segmentation process in vertebrates.


Assuntos
Relógios Biológicos/fisiologia , Padronização Corporal/fisiologia , Embrião de Mamíferos/embriologia , Embrião não Mamífero , Proteínas/metabolismo , Somitos/metabolismo , Coluna Vertebral/embriologia , Vertebrados/embriologia , Animais , Proteína Axina , Relógios Biológicos/genética , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Embrião de Mamíferos/metabolismo , Retroalimentação/fisiologia , Feto , Regulação da Expressão Gênica no Desenvolvimento/genética , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mesoderma/citologia , Mesoderma/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas/genética , Transdução de Sinais/genética , Somitos/citologia , Coluna Vertebral/citologia , Coluna Vertebral/metabolismo , Transativadores/genética , Transativadores/metabolismo , Vertebrados/metabolismo , Proteínas Wnt , Proteína Wnt3 , Proteína Wnt3A , beta Catenina
11.
Sci Rep ; 8(1): 11095, 2018 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-30038304

RESUMO

The numbers of thymic epithelial cells (TECs) and thymocytes steadily increase during embryogenesis. To examine this dynamic, we generated several TEC-specific transgenic mouse lines, which express fluorescent proteins in the nucleus, the cytosol and in the membranes under the control of the Foxn1 promoter. These tools enabled us to determine TEC numbers in tissue sections by confocal fluorescent microscopy, and in the intact organ by light-sheet microscopy. Compared to histological procedures, flow cytometric analysis of thymic cellularity is shown to underestimate the numbers of TECs by one order of magnitude; using enzymatic digestion of thymic tissue, the loss of cortical TECs (cTECs) is several fold greater than that of medullary TECs (mTECs), although different cTEC subsets appear to be still present in the final preparation. Novel reporter lines driven by Psmb11 and Prss16 promoters revealed the trajectory of differentiation of cTEC-like cells, and, owing to the additional facility of conditional cell ablation, allowed us to follow the recovery of such cells after their depletion during embryogenesis. Multiparametric histological analyses indicate that the new transgenic reporter lines not only reveal the unique morphologies of different TEC subsets, but are also conducive to the analysis of the complex cellular interactions in the thymus.


Assuntos
Epitélio/embriologia , Timo/embriologia , Animais , Comunicação Celular , Microambiente Celular , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Epitélio/metabolismo , Expressão Gênica , Genes Reporter , Camundongos Transgênicos , Células Estromais/citologia , Células Estromais/metabolismo , Timo/metabolismo
12.
Cell Rep ; 18(12): 2815-2824, 2017 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-28329675

RESUMO

Wnt/ß-catenin signaling is required for embryonic stem cell (ESC) pluripotency by inducing mesodermal differentiation and inhibiting neuronal differentiation; however, how ß-catenin counter-regulates these differentiation pathways is unknown. Here, we show that lysine 49 (K49) of ß-catenin is trimethylated (ß-catMe3) by Ezh2 or acetylated (ß-catAc) by Cbp. Significantly, ß-catMe3 acts as a transcriptional co-repressor of the neuronal differentiation genes sox1 and sox3, whereas ß-catAc acts as a transcriptional co-activator of the key mesodermal differentiation gene t-brachyury (t-bra). Furthermore, ß-catMe3 and ß-catAc are alternatively enriched on repressed or activated genes, respectively, during ESC and adult stem cell differentiation into neuronal or mesodermal progenitor cell lineages. Importantly, expression of a ß-catenin K49A mutant results in major defects in ESC differentiation. We conclude that ß-catenin K49 trimethylation and acetylation are key elements in regulating ESC pluripotency and differentiation potential.


Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Lisina/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , beta Catenina/química , beta Catenina/metabolismo , Acetilação , Animais , Anticorpos Monoclonais/metabolismo , Diferenciação Celular/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Regulação da Expressão Gênica , Metilação , Mutação/genética , Complexo Repressor Polycomb 2/metabolismo , Regiões Promotoras Genéticas/genética , Ratos Endogâmicos Lew
13.
Mech Dev ; 119(2): 127-35, 2002 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-12464426

RESUMO

Neural crest cells are essential for proper development of a variety of tissues and structures, including peripheral and autonomic nervous systems, facial skeleton, aortic arches and pharyngeal glands like the thymus and parathyroids. Previous work has shown that bone morphogenic protein (BMP) signalling is required for the production of migratory neural crest cells that contribute to the neurogenic and skeletogenic lineages. We show here that BMP-dependent neural crest cells are also required for development of the embryonic aortic arches and pharynx-derived glands. Blocking formation or migration of this crest cell population from the caudal hindbrain resulted in strong phenotypes in the cardiac outflow tract and the thymus. Thymic aplasia or hypoplasia occurs despite uncompromised gene induction in the pharyngeal endoderm. In addition, when hypoplastic thymic tissue is found, it is ectopically located, but functional in thymopoiesis. Our data indicate that thymic phenotypes produced by neural crest deficits result from aberrant formation of pharyngeal pouches and impaired migration of thymic primordia because the mesenchymal content in the branchial arches is below a threshold level.


Assuntos
Aorta Torácica/embriologia , Proteínas Morfogenéticas Ósseas/metabolismo , Crista Neural/embriologia , Animais , Animais Geneticamente Modificados , Linhagem da Célula , Movimento Celular , DNA Complementar/metabolismo , Síndrome de DiGeorge/genética , Regulação para Baixo , Biblioteca Gênica , Humanos , Hibridização In Situ , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Biológicos , Mutação , Crista Neural/metabolismo , Fenótipo , Transdução de Sinais , Timo/embriologia , Timo/metabolismo , Fatores de Tempo , Xenopus
14.
Mech Dev ; 120(12): 1423-32, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-14654215

RESUMO

We report that gene silencing via intracytoplasmic microinjections of morpholino-modified antisense oligonucleotides is an effective and reproducible method to study both maternal and zygotic gene functions during early and late stages of mouse preimplantation development. The zygotic expression of the beta-geo transgene in the ROSA26 mouse strain could be inhibited until at least the early blastula stages. Thus morpholino-triggered gene inactivation appears to be a useful method to study the functional role of genes in preimplantation development. Using this approach, we have investigated a potential role of maternal expression of Cdh1, the gene encoding the cell-adhesion molecule E-cadherin. Inhibition of translation of maternal E-cadherin mRNA causes a developmental arrest at the two-cell stage. BrUTP incorporation assays indicated that this developmental defect cannot be explained by a general failure in transcriptional activity. This defect is reversible since E-cadherin mRNA can rescue the affected embryos, suggesting that a functional adhesion complex, present at the junction between blastomeres, is a prerequisite for the normal development of the mouse preimplantation embryo. Our study thus reveals a previously unanticipated role of maternal E-cadherin during early stages of mouse development.


Assuntos
Caderinas/genética , Caderinas/metabolismo , Regulação para Baixo/genética , Desenvolvimento Embrionário e Fetal , Oligonucleotídeos Antissenso/genética , Animais , Adesão Celular , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Feminino , Camundongos , Oócitos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Gênica/genética , Transgenes/genética
15.
PLoS One ; 7(4): e35322, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22530006

RESUMO

Establishment of totipotency after somatic cell nuclear transfer (NT) requires not only reprogramming of gene expression, but also conversion of the cell cycle from quiescence to the precisely timed sequence of embryonic cleavage. Inadequate adaptation of the somatic nucleus to the embryonic cell cycle regime may lay the foundation for NT embryo failure and their reported lower cell counts. We combined bright field and fluorescence imaging of histone H(2b)-GFP expressing mouse embryos, to record cell divisions up to the blastocyst stage. This allowed us to quantitatively analyze cleavage kinetics of cloned embryos and revealed an extended and inconstant duration of the second and third cell cycles compared to fertilized controls generated by intracytoplasmic sperm injection (ICSI). Compared to fertilized embryos, slow and fast cleaving NT embryos presented similar rates of errors in M phase, but were considerably less tolerant to mitotic errors and underwent cleavage arrest. Although NT embryos vary substantially in their speed of cell cycle progression, transcriptome analysis did not detect systematic differences between fast and slow NT embryos. Profiling of amino acid turnover during pre-implantation development revealed that NT embryos consume lower amounts of amino acids, in particular arginine, than fertilized embryos until morula stage. An increased arginine supplementation enhanced development to blastocyst and increased embryo cell numbers. We conclude that a cell cycle delay, which is independent of pluripotency marker reactivation, and metabolic restraints reduce cell counts of NT embryos and impede their development.


Assuntos
Ciclo Celular/fisiologia , Reprogramação Celular/fisiologia , Aminoácidos/metabolismo , Animais , Arginina/metabolismo , Blastocisto/metabolismo , Análise por Conglomerados , Dano ao DNA , Transferência Embrionária , Embrião de Mamíferos/citologia , Embrião de Mamíferos/fisiologia , Desenvolvimento Embrionário/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Cinética , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Técnicas de Transferência Nuclear , Imagem com Lapso de Tempo , Transgenes
16.
Genesis ; 45(7): 456-9, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17607693

RESUMO

The trophectoderm (TE) of blastocysts, the first epithelium established in mammalian development, (1) plays signaling, supportive, and patterning functions during preimplantation development, (2) ensures embryo implantation into the uterine wall, and (3) gives rise to extraembryonic tissues essential for embryo patterning and growth after implantation. We show that mouse TE, itself permissive to lentiviral (LV) infection, represents a robust nonpermeable physical barrier to the virus particles, thereby shielding the cells of the inner cell mass from viral infection. This LV feature will allow modulations of gene expression in a lineage-specific manner, thus having significant applications in mouse functional genetics.


Assuntos
Linhagem da Célula , Técnicas de Transferência de Genes , Vetores Genéticos , Lentivirus , Animais , Feminino , Genes Reporter , Masculino , Camundongos , Camundongos Endogâmicos C57BL
17.
Development ; 134(1): 31-41, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17138661

RESUMO

During mammalian embryogenesis the trophectoderm represents the first epithelial structure formed. The cell adhesion molecule E-cadherin is ultimately necessary for the transition from compacted morula to the formation of the blastocyst to ensure correct establishment of adhesion junctions in the trophectoderm. Here, we analyzed to what extent E-cadherin confers unique adhesion and signaling properties in trophectoderm formation in vivo. Using a gene replacement approach, we introduced N-cadherin cDNA into the E-cadherin genomic locus. We show that the expression of N-cadherin driven from the E-cadherin locus reflects the expression pattern of endogenous E-cadherin. Heterozygous mice co-expressing E- and N-cadherin are vital and show normal embryonic development. Interestingly, N-cadherin homozygous mutant embryos phenocopy E-cadherin-null mutant embryos. Upon removal of the maternal E-cadherin, we demonstrate that N-cadherin is able to provide sufficient cellular adhesion to mediate morula compaction, but is insufficient for the subsequent formation of a fully polarized functional trophectoderm. When ES cells were isolated from N-cadherin homozygous mutant embryos and teratomas were produced, these ES cells differentiated into a large variety of tissue-like structures. Importantly, different epithelial-like structures expressing N-cadherin were formed, including respiratory epithelia, squamous epithelia with signs of keratinization and secretory epithelia with goblet cells. Thus, N-cadherin can maintain epithelia in differentiating ES cells, but not during the formation of the trophectoderm. Our results point to a specific and unique function for E-cadherin during mouse preimplantation development.


Assuntos
Blastocisto/fisiologia , Caderinas/genética , Caderinas/metabolismo , Ectoderma/fisiologia , Embrião de Mamíferos/metabolismo , Animais , Blastocisto/citologia , Caderinas/análise , Adesão Celular , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Cruzamentos Genéticos , Ectoderma/citologia , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia de Vídeo , beta-Galactosidase/metabolismo
18.
J Immunol ; 178(4): 2241-8, 2007 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-17277129

RESUMO

Endoplasmic reticulum-associated aminopeptidase 1 (ERAP1) is involved in the final processing of endogenous peptides presented by MHC class I molecules to CTLs. We generated ERAP1-deficient mice and analyzed cytotoxic responses upon infection with three viruses, including lymphocytic choriomeningitis virus, which causes vigorous T cell activation and is controlled by CTLs. Despite pronounced effects on the presentation of selected epitopes, the in vivo cytotoxic response was altered for only one of several epitopes tested. Moreover, control of lymphocytic choriomeningitis virus was not impaired in the knockout mice. Thus, we conclude that lack of ERAP1 has little influence on antiviral immunohierarchies and antiviral immunity in the infections studied. We also focused on the role of ERAP1 in cross-presentation. We demonstrate that ERAP1 is required for efficient cross-presentation of cell-associated Ag and of OVA/anti-OVA immunocomplexes. Surprisingly, however, ERAP1 deficiency has no effect on cross-presentation of soluble OVA, suggesting that for soluble exogenous proteins, final processing may not take place in an environment containing active ERAP1.


Assuntos
Aminopeptidases/imunologia , Apresentação de Antígeno/imunologia , Infecções por Arenaviridae/imunologia , Retículo Endoplasmático/imunologia , Vírus da Coriomeningite Linfocítica/imunologia , Linfócitos T Citotóxicos/imunologia , Aminopeptidases/deficiência , Animais , Apresentação de Antígeno/genética , Complexo Antígeno-Anticorpo/genética , Complexo Antígeno-Anticorpo/imunologia , Infecções por Arenaviridae/genética , Reações Cruzadas/genética , Reações Cruzadas/imunologia , Retículo Endoplasmático/genética , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Vírus da Coriomeningite Linfocítica/genética , Camundongos , Camundongos Knockout , Antígenos de Histocompatibilidade Menor
19.
Cell ; 125(5): 971-86, 2006 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-16751105

RESUMO

Vertebrate skeletogenesis involves two processes, skeletal patterning and osteoblast differentiation. Here, we show that Satb2, encoding a nuclear matrix protein, is expressed in branchial arches and in cells of the osteoblast lineage. Satb2-/- mice exhibit both craniofacial abnormalities that resemble those observed in humans carrying a translocation in SATB2 and defects in osteoblast differentiation and function. Multiple osteoblast-specific genes were identified as targets positively regulated by SATB2. In addition, SATB2 was found to repress the expression of several Hox genes including Hoxa2, an inhibitor of bone formation and regulator of branchial arch patterning. Molecular analysis revealed that SATB2 directly interacts with and enhances the activity of both Runx2 and ATF4, transcription factors that regulate osteoblast differentiation. This synergy was genetically confirmed by bone formation defects in Satb2/Runx2 and Satb2/Atf4 double heterozygous mice. Thus, SATB2 acts as a molecular node in a transcriptional network regulating skeletal development and osteoblast differentiation.


Assuntos
Anormalidades Craniofaciais/genética , Ossos Faciais/anormalidades , Proteínas de Ligação à Região de Interação com a Matriz/genética , Osteoblastos/metabolismo , Osteogênese/genética , Crânio/anormalidades , Fatores de Transcrição/genética , Fator 4 Ativador da Transcrição/genética , Animais , Padronização Corporal/genética , Região Branquial/anormalidades , Região Branquial/citologia , Região Branquial/metabolismo , Diferenciação Celular/genética , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Anormalidades Craniofaciais/metabolismo , Anormalidades Craniofaciais/fisiopatologia , Ossos Faciais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Genes Homeobox/genética , Proteínas de Homeodomínio/genética , Camundongos , Camundongos Knockout , Mutação/genética , Osteoblastos/citologia , Proteínas Repressoras/genética
20.
Dev Biol ; 291(2): 382-97, 2006 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-16458883

RESUMO

SOX9 is an evolutionary conserved transcription factor that is expressed in a variety of tissues, with essential functions in cartilage, testis, heart, glial cell, inner ear and neural crest development. By comparing human and pufferfish genomic sequences, we previously identified eight highly conserved sequence elements between 290 kb 5' and 450 kb 3' to human SOX9. In this study, we assayed the regulatory potential of elements E1 to E7 in transgenic mice using a lacZ reporter gene driven by a 529 bp minimal mouse Sox9 promoter. We found that three of these elements and the Sox9 promoter control distinct subsets of the tissue-specific expression pattern of Sox9. E3, located 251 kb 5' to SOX9, directs lacZ expression to cranial neural crest cells and to the inner ear. E1 is located 28 kb 5' to SOX9 and controls expression in the node, notochord, gut, bronchial epithelium and pancreas. Transgene expression in the neuroectoderm is mediated by E7, located 95 kb 3' to SOX9, which regulates expression in the telencephalon and midbrain, and by the Sox9 minimal promoter which controls expression in the ventral spinal cord and hindbrain. We show that E3-directed reporter gene expression in neural crest cells of the first but not of the second and third pharyngeal arch is dependent on beta-catenin, revealing a complex regulation of Sox9 in cranial neural crest cells. Moreover, we identify and discuss highly conserved transcription factor binding sites within enhancer E3 that are in good agreement with current models for neural crest and inner ear development. Finally, we identify enhancer E1 as a cis-regulatory element conserved between vertebrates and invertebrates, indicating that some cis-regulatory sequences that control developmental genes in vertebrates might be phylogenetically ancient.


Assuntos
Orelha Interna/embriologia , Elementos Facilitadores Genéticos/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Grupo de Alta Mobilidade/genética , Crista Neural/embriologia , Fatores de Transcrição/genética , Animais , Sequência de Bases , Sítios de Ligação , Encéfalo/embriologia , Encéfalo/metabolismo , Sequência Conservada , Orelha Interna/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Camundongos , Dados de Sequência Molecular , Crista Neural/metabolismo , Notocorda/metabolismo , Regiões Promotoras Genéticas , Fatores de Transcrição SOX9 , beta Catenina/fisiologia
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