RESUMEN
The forkhead box N1 (Foxn1) protein is the key regulator of thymic epithelial cell (TEC) development, yet how Foxn1 functions remains largely unknown. All mature TECs arise from Foxn1-expressing progenitors/immature TECs and it is widely assumed that TECs as a whole are defined by Foxn1 expression. However, data on the Foxn1 protein are virtually lacking. In this study, we developed novel tools to visualize Foxn1 protein expression at single-cell resolution. We generated Foxn1 knock-in mice expressing a C-terminal hemagglutinin-tagged Foxn1 protein, and a cytometry-grade monoclonal anti-Foxn1 Ab. We evaluated Foxn1 expression patterns in TEC subsets and its dynamics during normal thymus development, aging, injury, and regeneration. Upon challenges, upregulation of Foxn1 was a common feature of thymus regeneration, but the timing of Foxn1 expression changed and the responding TEC subsets depended on the type of treatment. Whereas dexamethasone and recombinant human fibroblast growth factor 7 promoted expansion of Foxn1(+)Ly51(+)CD80(-) TECs, castration led to expansion of Foxn1(+)Ly51(-)CD80(+) TECs. Collectively, Foxn1 expression is highly heterogeneous in the normal thymus, with large fractions of Foxn1(low) or Foxn1(-) TECs accumulating with age. Furthermore, Foxn1 expression is responsive to perturbations.
Asunto(s)
Células Epiteliales/fisiología , Factores de Transcripción Forkhead/metabolismo , Timo/fisiología , Envejecimiento/fisiología , Animales , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Dexametasona/farmacología , Células Epiteliales/efectos de los fármacos , Factor 7 de Crecimiento de Fibroblastos/farmacología , Factores de Transcripción Forkhead/genética , Regulación del Desarrollo de la Expresión Génica , Técnicas de Sustitución del Gen , Humanos , Ratones , Ratones Endogámicos C57BL , Regeneración/fisiologíaRESUMEN
Loss of tissue organization is a hallmark of the early stages of cancer, and there is considerable interest in proteins that maintain normal tissue architecture. Prostate epithelial cells cultured in Matrigel form three-dimensional acini that mimic aspects of prostate gland development. The organization of these structures requires the tumor suppressor Dickkopf-3 (Dkk-3), a divergent member of the Dkk family of secreted Wnt signalling antagonists that is frequently downregulated in prostate cancer. To gain further insight into the function of Dkk-3 in the prostate, we compared the prostates of Dkk3-null mice with those of control littermates. We found increased proliferation of prostate epithelial cells in the mutant mice and changes in prostate tissue organization. Consistent with these observations, cell proliferation was elevated in acini formed by human prostate epithelial cells stably silenced for Dkk-3. Silencing of Dkk-3 increased TGF-ß/Smad signalling, and inhibitors of TGF-ß/Smad signalling rescued the defective acinar phenotype caused by loss of Dkk-3. These findings suggest that Dkk-3 maintains the structural integrity of the prostate gland by limiting TGF-ß/Smad signalling.
Asunto(s)
Péptidos y Proteínas de Señalización Intercelular/metabolismo , Morfogénesis/fisiología , Próstata/citología , Próstata/metabolismo , Proteínas Smad/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Western Blotting , Línea Celular , Humanos , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intercelular/genética , Masculino , Ratones , Ratones Noqueados , Morfogénesis/genética , Proteínas Smad/genética , Factor de Crecimiento Transformador beta/genéticaRESUMEN
Male sex determination in mammals is induced by Sry, a gene whose regulation is poorly understood. Here we show that mice mutant for the stress-response gene Gadd45g display complete male-to-female sex reversal. Gadd45g and Sry have a strikingly similar expression pattern in the genital ridge, and they are coexpressed in gonadal somatic cells. In Gadd45g mutants, Sry expression is delayed and reduced, and yet Sry seemed to remain poised for expression, because its promoter is demethylated on schedule and is occupied by active histone marks. Instead, p38 MAPK signaling is impaired in Gadd45g mutants. Moreover, the transcription factor GATA4, which is required for Sry expression, binds to the Sry promoter in vivo in a MAPK-dependent manner. The results suggest that a signaling cascade, involving GADD45G â p38 MAPK â GATA4 â SRY, regulates male sex determination.
Asunto(s)
Proteínas Portadoras/metabolismo , Procesos de Determinación del Sexo/fisiología , Proteína de la Región Y Determinante del Sexo/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Proteínas Portadoras/genética , Metilación de ADN , Femenino , Factor de Transcripción GATA4/metabolismo , Regulación del Desarrollo de la Expresión Génica , Genes sry , Disgenesia Gonadal 46 XY/embriología , Disgenesia Gonadal 46 XY/genética , Disgenesia Gonadal 46 XY/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Sistema de Señalización de MAP Quinasas , Masculino , Ratones , Ratones Mutantes , Ratones Transgénicos , Factor de Transcripción SOX9/genética , Factor de Transcripción SOX9/metabolismo , Procesos de Determinación del Sexo/genética , Proteína de la Región Y Determinante del Sexo/genética , Testículo/embriología , Testículo/metabolismoRESUMEN
The vertebrate embryonic vasculature develops from angioblasts, which are specified from mesodermal precursors and develop in close association with blood cells. The signals that regulate embryonic vasculogenesis and angiogenesis are incompletely understood. Here, we show that R-spondin 3 (Rspo3), a member of a novel family of secreted proteins in vertebrates that activate Wnt/beta-catenin signaling, plays a key role in these processes. In Xenopus embryos, morpholino antisense knockdown of Rspo3 induces vascular defects because Rspo3 is essential for regulating the balance between angioblast and blood cell specification. In mice, targeted disruption of Rspo3 leads to embryonic lethality caused by vascular defects. Specifically in the placenta, remodeling of the vascular plexus is impaired. In human endothelial cells, R-spondin signaling promotes proliferation and sprouting angiogenesis in vitro, indicating that Rspo3 can regulate endothelial cells directly. We show that vascular endothelial growth factor is an immediate early response gene and a mediator of R-spondin signaling. The results identify Rspo3 as a novel, evolutionarily conserved angiogenic factor in embryogenesis.
Asunto(s)
Células Sanguíneas/citología , Diferenciación Celular , Neovascularización Fisiológica , Transducción de Señal , Trombospondinas/metabolismo , Proteínas Wnt/metabolismo , Proteínas de Xenopus/metabolismo , Animales , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Embrión no Mamífero/embriología , Embrión no Mamífero/metabolismo , Células Endoteliales/citología , Femenino , Regulación del Desarrollo de la Expresión Génica , Hematopoyesis , Ratones , Placenta/metabolismo , Trombospondinas/genética , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Proteínas Wnt/genética , Proteínas de Xenopus/genética , Xenopus laevis/embriología , Xenopus laevis/genética , Xenopus laevis/metabolismo , beta Catenina/metabolismoRESUMEN
Kremen1 and Kremen2 (Krm1 and Krm2) are transmembrane coreceptors for Dickkopf1 (Dkk1), an antagonist of Wnt/beta-catenin signaling. The physiological relevance of Kremen proteins in mammals as Wnt modulators is unresolved. We generated and characterized Krm mutant mice and found that double mutants show enhanced Wnt signaling accompanied by ectopic postaxial forelimb digits and expanded apical ectodermal ridges. Triple mutant Krm1(-/-) Krm2(-/-) Dkk1(+/-) mice show enhanced growth of ectopic digits, indicating that Dkk1 and Krm genes genetically interact during limb development. Wnt/beta-catenin signaling also plays a critical role in bone formation. Single Krm mutants show normal bone formation and bone mass, while double mutants show increased bone volume and bone formation parameters. Our study provides the first genetic evidence for a functional interaction of Kremen proteins with Dkk1 as negative regulators of Wnt/beta-catenin signaling and reveals that Kremen proteins are not universally required for Dkk1 function.
Asunto(s)
Densidad Ósea/genética , Eliminación de Gen , Marcación de Gen , Deformidades Congénitas de las Extremidades/genética , Proteínas de la Membrana/genética , Proteínas Wnt/metabolismo , Animales , Tipificación del Cuerpo , Línea Celular , Embrión de Mamíferos/anomalías , Embrión de Mamíferos/patología , Extremidades/embriología , Extremidades/patología , Fertilidad , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Deformidades Congénitas de las Extremidades/embriología , Deformidades Congénitas de las Extremidades/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Osteogénesis , Unión Proteica , Transducción de Señal , Trombospondinas/metabolismoRESUMEN
DNA methylation is an epigenetic modification that is essential for gene silencing and genome stability in many organisms. Although methyltransferases that promote DNA methylation are well characterized, the molecular mechanism underlying active DNA demethylation is poorly understood and controversial. Here we show that Gadd45a (growth arrest and DNA-damage-inducible protein 45 alpha), a nuclear protein involved in maintenance of genomic stability, DNA repair and suppression of cell growth, has a key role in active DNA demethylation. Gadd45a overexpression activates methylation-silenced reporter plasmids and promotes global DNA demethylation. Gadd45a knockdown silences gene expression and leads to DNA hypermethylation. During active demethylation of oct4 in Xenopus laevis oocytes, Gadd45a is specifically recruited to the site of demethylation. Active demethylation occurs by DNA repair and Gadd45a interacts with and requires the DNA repair endonuclease XPG. We conclude that Gadd45a relieves epigenetic gene silencing by promoting DNA repair, which erases methylation marks.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Metilación de ADN , Reparación del ADN , Epigénesis Genética , Proteínas Nucleares/metabolismo , Regulación hacia Arriba/genética , Proteínas de Xenopus/metabolismo , Xenopus/genética , Xenopus/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Línea Celular , Clonación Molecular , Silenciador del Gen , Genes Reporteros/genética , Humanos , Ratones , Proteínas Nucleares/genética , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Oocitos/metabolismo , Regiones Promotoras Genéticas/genética , Especificidad por Sustrato , Proteínas de Xenopus/genéticaRESUMEN
Animal cloning by nuclear transplantation in amphibia was demonstrated almost half a century ago and raised the question of the mechanisms and genes involved in nuclear reprogramming. Here, we demonstrate nuclear reprogramming of permeabilized human cells using extracts from Xenopus laevis eggs and early embryos. We show upregulation of pluripotency markers Oct-4 and germ cell alkaline phosphatase (GCAP) in 293T cells and human primary leukocytes. Reprogrammed leukocytes had a limited life span and did not express surface antigens characteristic of pluripotent cells, indicating that reprogramming was incomplete. Reprogramming activity was detected in egg and early embryo extracts until early blastula stage. Late blastula-stage extracts were not only inactive but also inhibitory to reprogramming. Screening for factors required for reprogramming identified the chromatin remodeling ATPase BRG1. Antibody depletion of BRG1 protein or expression of dominant-negative BRG1 abolished the reprogramming ability of amphibian extracts. Conversely, overexpression of BRG1 in Xenopus animal caps extended their competence from blastula to gastrula stage to respond to basic fibroblast growth factor (bFGF) treatment with induction of the mesodermal marker Xbra. Dissection of the molecular machinery using a simplified assay system may aid in achieving complete nuclear reprogramming of somatic cells for regenerative medicine.