RESUMEN
The gene trap method for embryonic stem cells is an efficient method for identifying new genes that are involved in development. Using this method, we identified a novel gene called helicase family gene related to gastrulation (helG). Helicase family proteins regulate many systems in the body that are related to cell survival. HelG encodes a protein of 137 kDa, which contains a DExH helicase motif that is now named DHX30. HelG is strongly expressed in neural cells (ie, in the headfold, neural plate, neural tube, and brain) and somites during embryogenesis. Growing homozygous mutant embryos have neither differentiated somites nor brains. In these mutants, development was retarded by embryonic day 7.5 (E7.5), and the mutants died at E9.5. After the purification of HelG, an untwisting experiment was performed to confirm the helicase activity of HelG for DNA in vitro. We report for the first time that a helicase family gene is required for differentiation during embryogenesis; this gene might interact with polynucleotides to regulate some genes that are important for early development and has a structure similar to that of a human DExH box helicase.
Asunto(s)
Desarrollo Embrionario/genética , Células Madre Embrionarias/enzimología , Gastrulación/genética , Regulación del Desarrollo de la Expresión Génica , ARN Helicasas/genética , Secuencia de Aminoácidos , Animales , ADN Complementario/genética , Inducción Enzimática , Genes Letales , Genes Reporteros , Genes Sintéticos , Vectores Genéticos/genética , Estratos Germinativos/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , ARN Helicasas/biosíntesis , ARN Helicasas/química , ARN Helicasas/deficiencia , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
BACKGROUND: Voltage-dependent block of the NMDA receptor by Mg2+ is thought to be central to the unique involvement of this receptor in higher brain functions. However, the in vivo role of the Mg2+ block in the mammalian brain has not yet been investigated, because brain-wide loss of the Mg2+ block causes perinatal lethality. In this study, we used a brain-region specific knock-in mouse expressing an NMDA receptor that is defective for the Mg2+ block in order to test its role in neural information processing. RESULTS: We devised a method to induce a single amino acid substitution (N595Q) in the GluN2A subunit of the NMDA receptor, specifically in the hippocampal dentate gyrus in mice. This mutation reduced the Mg2+ block at the medial perforant path-granule cell synapse and facilitated synaptic potentiation induced by high-frequency stimulation. The mutants had more stable hippocampal place fields in the CA1 than the controls did, and place representation showed lower sensitivity to visual differences. In addition, behavioral tests revealed that the mutants had a spatial working memory deficit. CONCLUSIONS: These results suggest that the Mg2+ block in the dentate gyrus regulates hippocampal spatial information processing by attenuating activity-dependent synaptic potentiation in the dentate gyrus.
Asunto(s)
Giro Dentado/metabolismo , Magnesio/farmacología , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Envejecimiento/fisiología , Animales , Secuencia de Bases , Giro Dentado/efectos de los fármacos , Giro Dentado/patología , Giro Dentado/fisiopatología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Marcación de Gen , Integrasas/metabolismo , Ratones , Ratones Mutantes Neurológicos , Datos de Secuencia Molecular , Proteínas Mutantes/metabolismo , Neurogénesis/efectos de los fármacos , Plasticidad Neuronal/efectos de los fármacos , Especificidad de Órganos/efectos de los fármacos , Subunidades de Proteína/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismoRESUMEN
Polypyrimidine tract-binding protein (PTB) is a well-characterized RNA-binding protein and known to be preferentially expressed in neural stem cells (NSCs) in the central nervous system; however, its role in NSCs in the developing brain remains unclear. To explore the role of PTB in embryonic NSCs in vivo, Nestin-Cre-mediated conditional Ptb knockout mice were generated for this study. In the mutant forebrain, despite the depletion of PTB protein, neither abnormal neurogenesis nor flagrant morphological abnormalities were observed at embryonic day 14.5 (E14.5). Nevertheless, by 10 weeks, nearly all mutant mice succumbed to hydrocephalus (HC), which was caused by a lack of the ependymal cell layer in the dorsal cortex. Upon further analysis, a gradual loss of adherens junctions (AJs) was observed in the ventricular zone (VZ) of the dorsal telencephalon in the mutant brains, beginning at E14.5. In the AJs-deficient VZ, impaired interkinetic nuclear migration and precocious differentiation of NSCs were observed after E14.5. These findings demonstrated that PTB depletion in the dorsal telencephalon is causally involved in the development of HC and that PTB is important for the maintenance of AJs in the NSCs of the dorsal telencephalon.
Asunto(s)
Uniones Adherentes/ultraestructura , Hidrocefalia/etiología , Proteína de Unión al Tracto de Polipirimidina/fisiología , Telencéfalo/embriología , Animales , Hidrocefalia/genética , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Células-Madre Neurales/ultraestructura , Proteína de Unión al Tracto de Polipirimidina/genética , Telencéfalo/anomalíasRESUMEN
Methyl CpG-binding protein 2 gene (MeCP2) mutations are implicated in Rett syndrome (RTT), one of the common causes of female mental retardation. Two MeCP2 isoforms have been reported: MeCP2_e2 (splicing of all four exons) and MeCP2_e1 (alternative splicing of exons 1, 3, and 4). Their relative expression levels vary among tissues, with MeCP2_e1 being more dominant in adult brain, whereas MeCP2_e2 is expressed more abundantly in placenta, liver, and skeletal muscle. In this study, we performed specific disruption of the MeCP2_e2-defining exon 2 using the Cre-loxP system and examined the consequences of selective loss of MeCP2_e2 function in vivo. We performed behavior evaluation, gene expression analysis, using RT-PCR and real-time quantitative PCR, and histological analysis. We demonstrate that selective deletion of MeCP2_e2 does not result in RTT-associated neurological phenotypes but confers a survival disadvantage to embryos carrying a MeCP2_e2 null allele of maternal origin. In addition, we reveal a specific requirement for MeCP2_e2 function in extraembryonic tissue, where selective loss of MeCP2_e2 results in placenta defects and up-regulation of peg-1, as determined by the parental origin of the mutant allele. Taken together, our findings suggest a novel role for MeCP2 in normal placenta development and illustrate how paternal X chromosome inactivation in extraembryonic tissues confers a survival disadvantage for carriers of a mutant maternal MeCP2_e2 allele. Moreover, our findings provide an explanation for the absence of reports on MeCP2_e2-specific exon 2 mutations in RTT. MeCP2_e2 mutations in humans may result in a phenotype that evades a diagnosis of RTT.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Proteína 2 de Unión a Metil-CpG/química , Alelos , Empalme Alternativo , Animales , Apoptosis , Supervivencia Celular , Modelos Animales de Enfermedad , Epigénesis Genética , Femenino , Proteína 2 de Unión a Metil-CpG/metabolismo , Ratones , Fenotipo , Placenta/metabolismo , Placenta/fisiología , Embarazo , Unión Proteica , Isoformas de Proteínas , Síndrome de Rett/genética , Síndrome de Rett/metabolismoRESUMEN
The generation of mice lacking the expression of the IRF3 transcription factor (Irf3(-/-) mice) has revealed its crucial role in the activation of the type I IFN response. The Bcl2l12 gene, encoding Bcl2L12 protein structurally related to the Bcl-2 family, was found to almost overlap with the Irf3 gene, and the null mutation previously introduced into the Irf3 allele resulted in the functional inactivation of the Bcl2l12 gene; therefore, the mice are correctly termed Irf3(-/-)Bcl2l12(-/-) mice. Embryonic fibroblasts from Irf3(-/-)Bcl2l12(-/-) mice (Irf3(-/-)Bcl2l12(-/-) MEFs) showed resistance to DNA damage-induced apoptosis, accompanied by impaired caspase cleavage. This apoptotic defect in Irf3(-/-)Bcl2l12(-/-) MEFs was rescued by the ectopic expression of Bcl2L12, but not IRF3. The Bcl2L12-mediated apoptotic response depended on the cell type and extracellular stimulus. In contrast, the previously reported defect in the induction of type I IFN genes by nucleic acids in Irf3(-/-)Bcl2l12(-/-) MEFs was rescued by expressing IRF3, but not Bcl2L12. Thus, our present study revealed, on the one hand, a cell type-dependent proapoptotic function of Bcl2L12 and, on the other hand, confirmed the essential role of IRF3 in type I IFN response.
Asunto(s)
Apoptosis , Factor 3 Regulador del Interferón/metabolismo , Mutación , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Western Blotting , Caspasas/metabolismo , Células Cultivadas , Daño del ADN , Fibroblastos/citología , Fibroblastos/metabolismo , Factor 3 Regulador del Interferón/genética , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Timo/citología , Timo/metabolismo , TransfecciónRESUMEN
Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that is widely expressed in the brain, and plays key roles in various cellular processes in response to both extracellular and intracellular stimuli. Here, we explored the role of FAK in cerebellar development. In the mouse cerebellum, FAK was found to be distributed as tiny cytoplasmic aggregates in various neuronal and glial elements, including Purkinje cells (PCs), Bergmann glia (BG), parallel fiber (PF)-terminals and climbing fiber (CF)-terminals. The neuron/glia-specific ablation of FAK impaired cerebellar foliation, such as variable decreases in foliation sizes and the lack of intercrural and precentral fissures. Some of the BG cells became situated ectopically in the molecular layer. Furthermore, the FAK ablation altered the innervation territories of CFs and PFs on PCs. CF innervation regressed to the basal portion of proximal dendrites and somata, whereas ectopic spines protruded from proximal dendrites and PFs expanded their territory by innervating the ectopic spines. Furthermore, the persistence of surplus CFs innervating PC somata caused multiple innervation. When FAK was selectively ablated in PCs, diminished dendritic innervation and persistent somatic innervation by CFs were observed, whereas cerebellar foliation and cell positioning of BG were normally retained. These results suggest that FAK in various neuronal and glial elements is required for the formation of normal histoarchitecture and cytoarchitecture in the cerebellum, and for the construction of proper innervation territory and synaptic wiring in PCs.
Asunto(s)
Cerebelo/enzimología , Cerebelo/ultraestructura , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Neuroglía/ultraestructura , Neuronas/ultraestructura , Animales , Técnica del Anticuerpo Fluorescente , Proteína-Tirosina Quinasas de Adhesión Focal/genética , Expresión Génica , Procesamiento de Imagen Asistido por Computador , Hibridación in Situ , Ratones , Ratones Mutantes , Microscopía Confocal , Neuroglía/metabolismo , Neuronas/metabolismo , ARN Mensajero/análisisRESUMEN
Although platelet-derived growth factor (PDGF)-BB activates PDGF receptor-beta (PDGFR-beta) and, in turn, inhibits the glutamate N-methyl-D-aspartate (NMDA) receptor function, whether PDGF-BB modulates the CNS function mediated by another glutamate receptors, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors, remains poorly understood. Here we now report the inhibitory effect of PDGF-BB on the AMPA receptor function in the nucleus tractus solitarius (NTS) by using slice patch-clamp techniques. Excitatory postsynaptic currents (EPSCs) were evoked by electrical stimulation of the tractus solitarius in mouse NTS second-order neurons. EPSCs were nearly completely eliminated by CNQX but not by MK-801, implying mediation through non-NMDA receptors. PDGF-BB significantly decreased the amplitude of EPSCs without affecting the mean decay time constant. This inhibitory effect was transient and reversible after removing PDGF-BB. Furthermore, PDGF-BB significantly reduced the amplitude of AMPA-induced currents in NTS neurons, which showed that PDGF-BB could suppress the AMPA receptor-mediated excitatory input via the postsynaptic mechanism. The inhibitory effect of PDGF-BB on EPSCs was not observed in mutant mice with conditional deletion of the PDGFR-beta gene in neurons. Together, these studies suggest that the PDGF-B/PDGFR-beta axis inhibits the AMPA receptor-mediated synaptic transmission that comprises the major part of the primary afferent to the NTS second-order neuron. The detected inhibitory action may be involved in the CNS regulation of the respiratory response.
Asunto(s)
Inductores de la Angiogénesis/farmacología , Factor de Crecimiento Derivado de Plaquetas/farmacología , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/fisiología , Receptores AMPA/fisiología , Núcleo Solitario/citología , Transmisión Sináptica/efectos de los fármacos , 6-Ciano 7-nitroquinoxalina 2,3-diona/farmacología , Análisis de Varianza , Animales , Becaplermina , Maleato de Dizocilpina/farmacología , Interacciones Farmacológicas , Estimulación Eléctrica/métodos , Antagonistas de Aminoácidos Excitadores/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Potenciales Postsinápticos Excitadores/efectos de la radiación , Técnicas In Vitro , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Técnicas de Placa-Clamp/métodos , Monoéster Fosfórico Hidrolasas/metabolismo , Proteínas Proto-Oncogénicas c-sis , Tiempo de Reacción/efectos de los fármacos , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/deficiencia , Transmisión Sináptica/fisiología , Transmisión Sináptica/efectos de la radiaciónRESUMEN
In developing mammalian telencephalon, the loss of adherens junctions and cell cycle exit represent crucial steps in the differentiation of neuroepithelial cells into neurons, but the relationship between these cellular events remains obscure. Atypical protein kinase C (aPKC) is known to contribute to junction formation in epithelial cells and to cell fate determination for Drosophila neuroblasts. To elucidate the functions of aPKClambda, one out of two aPKC members, in mouse neocortical neurogenesis, a Nestin-Cre mediated conditional gene targeting system was employed. In conditional aPKClambda knockout mice, neuroepithelial cells of the neocortical region lost aPKClambda protein at embryonic day 15 and demonstrated a loss of adherens junctions, retraction of apical processes and impaired interkinetic nuclear migration that resulted in disordered neuroepithelial tissue architecture. These results are evidence that aPKClambda is indispensable for the maintenance of adherens junctions and may function in the regulation of adherens junction integrity upon differentiation of neuroepithelial cells into neurons. In spite of the loss of adherens junctions in the neuroepithelium of conditional aPKClambda knockout mice, neurons were produced at a normal rate. Therefore, we concluded that, at least in the later stages of neurogenesis, regulation of cell cycle exit is independent of adherens junctions.