RESUMEN
The olfactory tubercle (OT) is a striatal region that receives olfactory inputs. mRNAs of prodynorphin (Pdyn) and preproenkephalin (Penk), precursors of dynorphins and enkephalins, respectively, are strongly expressed in the striatum. Both produce opioid peptides with various physiological effects such as pain relief and euphoria. Recent studies have revealed that OT has anatomical and cytoarchitectonic domains that play different roles in odor-induced motivated behavior. Neuronal subtypes of the OT can be distinguished by their expression of the dopamine receptors D1 (Drd1) and D2 (Drd2). Here, we addressed whether and which type of opioid peptide precursors the D1- and D2-expressing neurons in the OT express. We used multiple fluorescence in situ hybridization for mRNAs of the opioid precursors and dopamine receptors to characterize mouse OT neurons. Pdyn was mainly expressed by Drd1-expressing cells in the dense cell layer (DCL) of the OT, whereas Penk was expressed primarily by Drd2-expressing cells in the DCL. We also confirmed the presence of a larger population of Pdyn-Penk-Drd1 co-expressing cells in the DCL of the anteromedial OT compared with the anterolateral OT. These observations will help understand whether and how dynorphins and enkephalins in the OT are involved in diverse odor-induced motivated behaviors.
Asunto(s)
Dinorfinas , Encefalinas , Neuronas/metabolismo , Tubérculo Olfatorio/citología , Precursores de Proteínas , Animales , Cuerpo Estriado/metabolismo , Dinorfinas/análisis , Dinorfinas/genética , Dinorfinas/metabolismo , Encefalinas/análisis , Encefalinas/genética , Encefalinas/metabolismo , Hibridación Fluorescente in Situ , Ratones , Tubérculo Olfatorio/metabolismo , ARN Mensajero/metabolismo , Receptores de Dopamina D1/metabolismoRESUMEN
Coordination of skilled movements and motor planning relies on the formation of regionally restricted brain circuits that connect cortex with subcortical areas during embryonic development. Layer 5 neurons that are distributed across most cortical areas innervate the pontine nuclei (basilar pons) by protrusion and extension of collateral branches interstitially along their corticospinal extending axons. Pons-derived chemotropic cues are known to attract extending axons, but molecules that regulate collateral extension to create regionally segregated targeting patterns have not been identified. Here, we discovered that EphA7 and EfnA5 are expressed in the cortex and the basilar pons in a region-specific and mutually exclusive manner, and that their repulsive activities are essential for segregating collateral extensions from corticospinal axonal tracts in mice. Specifically, EphA7 and EfnA5 forward and reverse inhibitory signals direct collateral extension such that EphA7-positive frontal and occipital cortical areas extend their axon collaterals into the EfnA5-negative rostral part of the basilar pons, whereas EfnA5-positive parietal cortical areas extend their collaterals into the EphA7-negative caudal part of the basilar pons. Together, our results provide a molecular basis that explains how the corticopontine projection connects multimodal cortical outputs to their subcortical targets.SIGNIFICANCE STATEMENT Our findings put forward a model in which region-to-region connections between cortex and subcortical areas are shaped by mutually exclusive molecules to ensure the fidelity of regionally restricted circuitry. This model is distinct from earlier work showing that neuronal circuits within individual cortical modalities form in a topographical manner controlled by a gradient of axon guidance molecules. The principle that a shared molecular program of mutually repulsive signaling instructs regional organization-both within each brain region and between connected brain regions-may well be applicable to other contexts in which information is sorted by converging and diverging neuronal circuits.
Asunto(s)
Orientación del Axón/fisiología , Efrina-A5/metabolismo , Neocórtex/embriología , Vías Nerviosas/embriología , Puente/embriología , Receptor EphA7/metabolismo , Animales , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Neocórtex/metabolismo , Vías Nerviosas/metabolismo , Puente/patologíaRESUMEN
Na,K-ATPase is a ubiquitous molecule contributing to the asymmetrical distribution of Na+ and K+ ions across the plasma membrane and maintenance of the membrane potential, a prerequisite of neuronal activity. Na,K-ATPase comprises three subunits (α, ß, and FXYD). The α subunit has four isoforms in mice, with three of them (α1, α2, and α3) expressed in the brain. However, the functional and biological significances of the different brain isoforms remain to be fully elucidated. Recent studies have revealed the association of Atp1a3, a gene encoding α3 subunit, with neurological disorders. To map the cellular distributions of the α subunit isoforms and their coexpression patterns, we evaluated the mRNA expression of Atp1a1, Atp1a2, and Atp1a3 by in situ hybridization in the mouse brain. Atp1a1 and Atp1a3 were expressed in neurons, whereas Atp1a2 was almost exclusively expressed in glial cells. Most neurons coexpressed Atp1a1 and Atp1a3, with highly heterogeneous expression levels across the brain regions and neuronal subtypes. We identified parvalbumin (PV)-expressing GABAergic neurons in the hippocampus, somatosensory cortex, and retrosplenial cortex as an example of a neuronal subtype expressing low Atp1a1 and high Atp1a3. The expression of Atp1b isoforms was also heterogeneous across brain regions and cellular subtypes. The PV-expressing neurons expressed a high level of Atp1b1 and a low level of Atp1b2 and Atp1b3. These findings provide basic information on the region- and neuronal-subtype-dependent expression of Na,K-ATPase α and ß subunit isoforms, as well as a rationale for the selective involvement of neurons expressing high levels of Atp1a3 in neurological disorders.
Asunto(s)
Encéfalo/anatomía & histología , Encéfalo/metabolismo , Neuronas/clasificación , Neuronas/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Encéfalo/citología , Masculino , Ratones , Isoformas de Proteínas/análisis , Isoformas de Proteínas/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/análisisRESUMEN
Collagen is the most abundant protein in the animal kingdom and has a unique triple-helical structure. It not only provides mechanical strength to tissues, but also performs specific biological functions as a multifaceted signaling molecule. Animal-derived collagen is therefore widely used as a biocompatible material in vitro and in vivo. In this study, we developed a novel peptide-based material that mimicked both the polymeric properties and a selected biological function of native collagen. This material was prepared by end-to-end multiple disulfide cross-linking of chemically synthesized triple-helical peptides. The peptide polymer showed a gel-forming property, and receptor-specific cell binding was observed in vitro by incorporating a peptide harboring an integrin α2ß1-binding sequence. Furthermore, cell signaling activity and biodegradability were tunable according to the polymer contents. The results demonstrated the potential of this material as a designer collagen. STATEMENT OF SIGNIFICANCE: Collagen is a useful biomaterial with the gel-forming property. It also exhibits various biological activities through the interaction of specific amino acid sequences displayed on the triple helix with functional biomacromolecules. Here we report a novel synthetic material, artificial collagen, by end-to-end cross-linking of chemically synthesized collagen-like triple-helical peptides. The material allows independent regulation of polymer properties, i.e. gel stiffness, and sequence-specific bioactivities by altering peptide compositions. This material can also be variously shaped, for example, thin films with high transparency. In addition, it has low inflamatogenic properties and tunable biodegradability in vivo.
Asunto(s)
Materiales Biomiméticos/química , Colágeno/química , Reactivos de Enlaces Cruzados/química , Disulfuros/química , Hidrogeles/química , Oligopéptidos/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Adhesión Celular , Línea Celular , Proliferación Celular , Módulo de Elasticidad , Matriz Extracelular/metabolismo , Humanos , Hidrogeles/metabolismo , Integrina alfa2beta1/química , Masculino , Ratones Endogámicos C57BL , Unión Proteica , Reología , Propiedades de SuperficieRESUMEN
The essential involvement of phosphoinositides in synaptic plasticity is well-established, but incomplete knowledge of the downstream molecular entities prevents us from understanding their signalling cascades completely. Here, we determined that Phldb2, of which pleckstrin-homology domain is highly sensitive to PIP3, functions as a phosphoinositide-signalling mediator for synaptic plasticity. BDNF application caused Phldb2 recruitment toward postsynaptic membrane in dendritic spines, whereas PI3K inhibition resulted in its reduced accumulation. Phldb2 bound to postsynaptic scaffolding molecule PSD-95 and was crucial for localization and turnover of PSD-95 in the spine. Phldb2 also bound to GluA1 and GluA2. Phldb2 was indispensable for the interaction between NMDA receptors and CaMKII, and the synaptic density of AMPA receptors. Therefore, PIP3-responsive Phldb2 is pivotal for induction and maintenance of LTP. Memory formation was impaired in our Phldb2-/- mice.
Asunto(s)
Proteínas Portadoras/metabolismo , Homólogo 4 de la Proteína Discs Large/metabolismo , Potenciación a Largo Plazo/fisiología , Proteínas de la Membrana/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Receptores AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animales , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Memoria , Ratones , Plasticidad Neuronal , Unión Proteica/fisiologíaRESUMEN
The neuronal spine is a small, actin-rich dendritic or somatic protrusion that serves as the postsynaptic compartment of the excitatory synapse. The morphology of the spine reflects the activity of the synapse and is regulated by the dynamics of the actin cytoskeleton inside, which is controlled by actin binding proteins such as non-muscle myosin. Previously, we demonstrated that the subcellular localization and function of myosin IIb are regulated by its binding partner, filamin-A interacting protein (FILIP). However, how the subcellular distribution of myosin IIb is controlled by FILIP is not yet known. The objective of this study was to identify potential binding partners of FILIP that contribute to its regulation of non-muscle myosin IIb. Pull-down assays detected a 70-kDa protein that was identified by mass spectrometry to be the chaperone protein Hsc70. The binding of Hsc70 to FILIP was controlled by the adenosine triphosphatase (ATPase) activity of Hsc70. Further, FILIP bound to Hsc70 via a domain that was not required for binding non-muscle myosin IIb. Inhibition of ATPase activity of Hsc70 impaired the effect of FILIP on the subcellular distribution of non-muscle myosin IIb. Further, in primary cultured neurons, an inhibitor of Hsc70 impeded the morphological change in spines induced by FILIP. Collectively, these results demonstrate that Hsc70 interacts with FILIP to mediate its effects on non-muscle myosin IIb and to regulate spine morphology.
Asunto(s)
Filaminas/metabolismo , Proteínas del Choque Térmico HSC70/metabolismo , Miosina Tipo IIB no Muscular/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/química , Adenosina Trifosfatasas/metabolismo , Animales , Células COS , Proteínas Portadoras/metabolismo , Células Cultivadas , Chlorocebus aethiops , Dendritas/metabolismo , Regulación de la Expresión Génica , Hipocampo/embriología , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Chaperonas Moleculares/metabolismo , Células 3T3 NIH , Neuronas/metabolismo , Corteza Piriforme/embriología , Unión Proteica , Ratas , Sinapsis/metabolismoRESUMEN
Learning and memory depend on morphological and functional changes to neural spines. Non-muscle myosin 2b regulates actin dynamics downstream of long-term potentiation induction. However, the mechanism by which myosin 2b is regulated in the spine has not been fully elucidated. Here, we show that filamin A-interacting protein (FILIP) is involved in the control of neural spine morphology and is limitedly expressed in the brain. FILIP bound near the ATPase domain of non-muscle myosin heavy chain IIb, an essential component of myosin 2b, and modified the function of myosin 2b by interfering with its actin-binding activity. In addition, FILIP altered the subcellular distribution of myosin 2b in spines. Moreover, subunits of the NMDA receptor were differently distributed in FILIP-expressing neurons, and excitation propagation was altered in FILIP-knockout mice. These results indicate that FILIP is a novel, region-specific modulator of myosin 2b.
Asunto(s)
Proteínas Portadoras/fisiología , Espinas Dendríticas/química , Espinas Dendríticas/metabolismo , Cadenas Pesadas de Miosina/metabolismo , Miosina Tipo IIB no Muscular/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Actinas/metabolismo , Animales , Western Blotting , Células Cultivadas , Técnicas para Inmunoenzimas , Inmunoprecipitación , Potenciación a Largo Plazo , Ratones , Ratones Endogámicos ICR , Ratones Noqueados , Neuronas/citología , Neuronas/metabolismo , Unión Proteica , RatasRESUMEN
Duchenne muscular dystrophy (DMD) is a devastating disease characterized by muscle wasting, loss of mobility and death in early adulthood. Satellite cells are muscle-resident stem cells responsible for the repair and regeneration of damaged muscles. One pathological feature of DMD is the progressive depletion of satellite cells, leading to the failure of muscle repair. Here, we attempted to explore the molecular mechanisms underlying satellite cell ablation in the dystrophin mutant mdx mouse, a well-established model for DMD. Initial muscle degeneration activates satellite cells, resulting in increased satellite cell number in young mdx mice. This is followed by rapid loss of satellite cells with age due to the reduced self-renewal ability of mdx satellite cells. In addition, satellite cell composition is altered even in young mdx mice, with significant reductions in the abundance of non-committed (Pax7+ and Myf5-) satellite cells. Using a Notch-reporter mouse, we found that the mdx satellite cells have reduced activation of Notch signaling, which has been shown to be necessary to maintain satellite cell quiescence and self-renewal. Concomitantly, the expression of Notch1, Notch3, Jag1, Hey1 and HeyL are reduced in the mdx primary myoblast. Finally, we established a mouse model to constitutively activate Notch signaling in satellite cells, and show that Notch activation is sufficient to rescue the self-renewal deficiencies of mdx satellite cells. These results demonstrate that Notch signaling is essential for maintaining the satellite cell pool and that its deficiency leads to depletion of satellite cells in DMD.
Asunto(s)
Envejecimiento/patología , Distrofia Muscular Animal/metabolismo , Distrofia Muscular Animal/patología , Receptores Notch/deficiencia , Células Satélite del Músculo Esquelético/metabolismo , Células Satélite del Músculo Esquelético/patología , Transducción de Señal , Animales , Recuento de Células , Proliferación Celular , Ratones Endogámicos mdx , Músculos/patología , Receptores Notch/metabolismoRESUMEN
BACKGROUND: The Wnts are secreted proteins that play important roles in skeletal myogenesis, muscle fiber type diversification, neuromuscular junction formation and muscle stem cell function. How Wnt proteins orchestrate such diverse activities remains poorly understood. Canonical Wnt signaling stabilizes ß-catenin, which subsequently translocate to the nucleus to activate the transcription of TCF/LEF family genes. METHODS: We employed TCF-reporter mice and performed analysis of embryos and of muscle groups. We further isolated fetal myoblasts and performed cell and molecular analyses. RESULTS: We found that canonical Wnt signaling is strongly activated during fetal myogenesis and weakly activated in adult muscles limited to the slow myofibers. Muscle-specific transgenic expression of a stabilized ß-catenin protein led to increased oxidative myofibers and reduced muscle mass, suggesting that canonical Wnt signaling promotes slow fiber types and inhibits myogenesis. By TCF-luciferase reporter assay, we identified Wnt-1 and Wnt-3a as potent activators of canonical Wnt signaling in myogenic progenitors. Consistent with in vivo data, constitutive overexpression of Wnt-1 or Wnt-3a inhibited the proliferation of both C2C12 and primary myoblasts. Surprisingly, Wnt-1 and Wnt-3a overexpression up-regulated BMP-4, and inhibition of BMP-4 by shRNA or recombinant Noggin protein rescued the myogenic inhibitory effect of Wnt-1 and Wnt-3a. Importantly, Wnt-3a or BMP-4 recombinant proteins promoted slow myosin heavy chain expression during myogenic differentiation of fetal myoblasts. CONCLUSIONS: These results demonstrate a novel interaction between canonical Wnt and BMP signaling that induces myogenic differentiation towards slow muscle phenotype.
RESUMEN
Glia-guided migration (glia-guided locomotion) during radial migration is a characteristic yet unique mode of migration. In this process, the directionality of migration is predetermined by glial processes and not by growth cones. Prior to the initiation of glia-guided migration, migrating neurons transform from multipolar to bipolar, but the molecular mechanisms underlying this multipolar-bipolar transition and the commencement of glia-guided migration are not fully understood. Here, we demonstrate that the multipolar-bipolar transition is not solely a cell autonomous event; instead, the interaction of growth cones with glial processes plays an essential role. Time-lapse imaging with lattice assays reveals the importance of vigorously active growth cones in searching for appropriate glial scaffolds, completing the transition, and initiating glia-guided migration. These growth cone activities are regulated by Abl kinase and Cdk5 via WAVE2-Abi2 through the phosphorylation of tyrosine 150 and serine 137 of WAVE2. Neurons that do not display such growth cone activities are mispositioned in a more superficial location in the neocortex, suggesting the significance of growth cones for the final location of the neurons. This process occurs in spite of the "inside-out" principle in which later-born neurons are situated more superficially.
Asunto(s)
Movimiento Celular/genética , Conos de Crecimiento/fisiología , Proteínas de Homeodominio/metabolismo , Neuroglía/fisiología , Neuronas/citología , Familia de Proteínas del Síndrome de Wiskott-Aldrich/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Factores de Edad , Animales , Cadherinas/metabolismo , Proliferación Celular , Células Cultivadas , Corteza Cerebral/citología , Chlorocebus aethiops , Sulfato de Dextran/metabolismo , Embrión de Mamíferos , Femenino , Regulación del Desarrollo de la Expresión Génica/genética , Técnicas de Transferencia de Gen , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de Homeodominio/genética , Inmunoprecipitación , Técnicas In Vitro , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Mutación/genética , Neuronas/fisiología , Embarazo , Interferencia de ARN/fisiología , Transfección , Familia de Proteínas del Síndrome de Wiskott-Aldrich/genéticaRESUMEN
Phosphatidylinositol 3,4,5-triphosphate (PtdIns(3,4,5)P(3)) accumulates at the leading edge of migrating cells and works, at least partially, as both a compass to indicate directionality and a hub for subsequent intracellular events. However, how PtdIns(3,4,5)P(3) regulates the migratory machinery has not been fully elucidated. Here, we demonstrate a novel mechanism for efficient lamellipodium formation that depends on PtdIns(3,4,5)P(3) and the reciprocal regulation of PtdIns(3,4,5)P(3) itself. LL5beta, whose subcellular localization is directed by membrane PtdIns(3,4,5)P(3), recruits the actin-cross-linking protein Filamin A to the plasma membrane, where PtdIns(3,4,5)P(3) accumulates, with the Filamin A-binding Src homology 2 domain-containing inositol polyphosphate 5-phosphatase 2 (SHIP2). A large and dynamic lamellipodium was formed in the presence of Filamin A and LL5beta by the application of epidermal growth factor. Conversely, depletion of either Filamin A or LL5beta or the overexpression of either an F-actin-cross-linking mutant of Filamin A or a mutant of LL5beta without its PtdIns(3,4,5)P(3)-interacting region inhibited such events in COS-7 cells. Because F-actin initially polymerizes near the plasma membrane, it is likely that membrane-recruited Filamin A efficiently cross-links newly polymerized F-actin, leading to enhanced lamellipodium formation at the site of PtdIns(3,4,5)P(3) accumulation. Moreover, we demonstrate that co-recruited SHIP2 dephosphorylates PtdIns(3,4,5)P(3) at the same location.
Asunto(s)
Proteínas Portadoras/metabolismo , Movimiento Celular/fisiología , Proteínas Contráctiles/metabolismo , Proteínas de Microfilamentos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Seudópodos/metabolismo , Actinas/genética , Actinas/metabolismo , Animales , Células COS , Proteínas Portadoras/genética , Membrana Celular/genética , Membrana Celular/metabolismo , Movimiento Celular/efectos de los fármacos , Chlorocebus aethiops , Proteínas Contráctiles/genética , Factor de Crecimiento Epidérmico/farmacología , Filaminas , Humanos , Proteínas de Microfilamentos/genética , Fosfatos de Fosfatidilinositol/genética , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas , Monoéster Fosfórico Hidrolasas/genética , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/fisiología , Seudópodos/genéticaRESUMEN
Satellite cells play a central role in mediating the growth and regeneration of skeletal muscle. However, whether satellite cells are stem cells, committed progenitors, or dedifferentiated myoblasts has remained unclear. Using Myf5-Cre and ROSA26-YFP Cre-reporter alleles, we observed that in vivo 10% of sublaminar Pax7-expressing satellite cells have never expressed Myf5. Moreover, we found that Pax7(+)/Myf5(-) satellite cells gave rise to Pax7(+)/Myf5(+) satellite cells through apical-basal oriented divisions that asymmetrically generated a basal Pax7(+)/Myf5(-) and an apical Pax7(+)/Myf5(+) cells. Prospective isolation and transplantation into muscle revealed that whereas Pax7(+)/Myf5(+) cells exhibited precocious differentiation, Pax7(+)/Myf5(-) cells extensively contributed to the satellite cell reservoir throughout the injected muscle. Therefore, we conclude that satellite cells are a heterogeneous population composed of stem cells and committed progenitors. These results provide critical insights into satellite cell biology and open new avenues for therapeutic treatment of neuromuscular diseases.
Asunto(s)
Músculo Esquelético/citología , Células Satélite del Músculo Esquelético/citología , Animales , Diferenciación Celular , Separación Celular , Trasplante de Células , Femenino , Proteínas Luminiscentes/metabolismo , Masculino , Ratones , Músculo Esquelético/metabolismo , Factor 5 Regulador Miogénico/metabolismo , Factor de Transcripción PAX7/metabolismo , Receptores Notch/metabolismo , Células Satélite del Músculo Esquelético/metabolismoRESUMEN
Notch/RBP-J signaling is required for generation of early T progenitors (ETP) and promotion of double-negative (DN) 4 cells from DN3 cells in thymocyte differentiation. However, whether Notch affects other steps during thymocyte differentiation remains unknown. Msx2-interacting nuclear target protein (Mint) is an endogenous inhibitor of Notch regulation. Concordantly, by ex vivo analyses of embryonic thymi and in vitro differentiation studies of fetal liver progenitors, we find that Mint deficiency enhances generation of ETP and DN4 cells. Unexpectedly, however, Mint deficiency impairs differentiation of ETP into DN2 cells, suggesting that Notch/RBP-J signaling negatively regulates DN1-DN2 transition.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Proteínas Nucleares/genética , Proteínas Nucleares/fisiología , Receptores Notch/metabolismo , Timo/citología , Animales , Diferenciación Celular , Cruzamientos Genéticos , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Biológicos , Proteínas de Unión al ARN , Transducción de Señal , Células Madre/citología , Timo/metabolismoRESUMEN
Successful implantation relies on precisely orchestrated and reciprocal signaling between the implanting blastocyst and the receptive uterus. We have examined the role of the Wnt/beta-catenin signaling pathway during the process of implantation and demonstrate that this pathway is activated during two distinct stages. Wnt/beta-catenin signaling is first transiently activated in circular smooth muscle forming a banding pattern of activity within the uterus on early day 4. Subsequently, activation is restricted to the luminal epithelium at the prospective site of implantation. Activation at both sites requires the presence of the blastocyst. Furthermore, inhibition of Wnt/beta-catenin signaling interferes with the process of implantation. Our results demonstrate that the Wnt/beta-catenin signaling pathway plays a central role in coordinating uterus-embryo interactions required for implantation.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Implantación del Embrión/fisiología , Embrión de Mamíferos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Transducción de Señal/fisiología , Transactivadores/metabolismo , Útero/fisiología , Animales , Epitelio/metabolismo , Estrógenos/metabolismo , Femenino , Ratones , Ratones Transgénicos , Embarazo , Útero/metabolismo , Proteínas Wnt , beta Catenina , beta-Galactosidasa/metabolismoRESUMEN
Gene duplication is a major genetic event that can produce multiple protein isoforms. Comparative sequence and functional analysis of related gene products can provide insights into protein family evolution. To characterize the Caenorhabditis elegans troponin I family, we analyzed gene structures, tissue expression patterns and RNAi phenotypes of four troponin I isoforms. Tissue expression patterns were determined using lacZ/gfp/rfp reporter gene assays. The tni-1, tni-2/unc-27 and tni-3 genes, each encoding a troponin I isoform, are uniquely expressed in body wall, vulval and anal muscles but at different levels; tni-4 was expressed solely in the pharynx. Expressing tni-1 and -2 gene RNAi caused motility defects similar to unc-27 (e155) mutant, a tni-2 null allele. The tni-3 RNAi expression produced egg laying defects while the tni-4 RNAi caused arrest at gastrulation. Overlay analyses were used to assay interactions between the troponin I and two troponin C isoforms. The three body wall troponin I isoforms interacted with body wall and pharyngeal troponin C isoforms; TNI-4 interacted only with pharyngeal troponin C. Our results suggest the body wall genes have evolved following duplication of the pharynx gene and provide important data about gene duplication and functional differentiation of nematode troponin I isoforms.
Asunto(s)
Caenorhabditis elegans/genética , Troponina C/metabolismo , Troponina I/genética , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans/metabolismo , Datos de Secuencia Molecular , Isoformas de Proteínas , Interferencia de ARN , Homología de Secuencia , Troponina C/genética , Troponina I/metabolismoRESUMEN
Notch is a transmembrane protein involved in cell fate determination. In the present study, we observed temporally and spatially restricted expression of Notch1 in developing cartilage. Notch1 was localized starting from the mesenchymal condensation stage of embryonic mouse forelimbs. Interestingly, although localization could not be detected in the proliferating chondrocytes, obvious immunoreactivity indicating its expression was retained in the perichondrial region. Next, we investigated the expression of Notch1 and related molecules in a chondrogenic cell line, ATDC5 cells. Notch1, Delta-like (Dll)1, Deltex2, and Deltex3 were coexpressed after 6-day insulin treatment. Expression of Hairy and Enhancer of split homologue (HES)-1 followed thereafter. These results suggest that Notch may have a role in the early stage of chondrogenesis. To assess the effect of Notch activation, we cultured ATDC5 cells with a myeloma clone constitutively expressing Dll1, a ligand of Notch. We also used an adenovirus vector to express the constitutively active Notch1 intracellular domain (NIC). Activating either the endogenous or exogenous Notch receptor dramatically inhibited chondrogenic cell differentiation of ATDC5 cells, as assessed by Alcian blue staining of the cells and chondrocyte differentiation markers. Last, we investigated the effect of NIC on the proliferation of the ATDC5 cells. Expression of NIC by the adenovirus strongly suppressed thymidine incorporation. These results indicate that Notch is expressed in the initial stage of chondrogenic cell differentiation and has a strong inhibitory effect on both differentiation and proliferation of the cells when activated. The expression of Notch decreases as chondrogenic differentiation proceeds; however, a population of the cells with sustained expression of Notch1 become perichondrial cells. Considering that the perichondrium acts as a stem cell source of osteoblasts and chondrocytes, Notch1 may have a role in the formation of these cells by suppressing both differentiation and proliferation.
Asunto(s)
Condrocitos/citología , Proteínas de la Membrana/metabolismo , Adenoviridae/genética , Adenoviridae/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Cartílago/citología , Diferenciación Celular , División Celular , Línea Celular , Condrocitos/metabolismo , Vectores Genéticos , Proteínas de Homeodominio/metabolismo , Proteínas de la Membrana/genética , Ratones , Receptores Notch , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Timidina/metabolismo , Factor de Transcripción HES-1RESUMEN
RBP-J is an essential signal mediator of all four Notches in nuclei. Loss-of-function analyses clearly show the crucial roles of RBP-J in commitment of T cells versus B cells as well as MZ B cells versus Fo B cells. Such Notch/RBP-J regulation of dichotomic differentiation steps in lymphocyte is reminiscent of the development of sensory organ precursors (SOPs) in Drosophila. Studies on RBP-J conditional knockout mice that have lost MZ B cells without affecting Fo B cell functions have shown that MZ B cells play pivotal roles in immune responses to blood-borne bacteria.
Asunto(s)
Linfocitos B/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Nucleares/metabolismo , Animales , Antígenos CD/metabolismo , Linfocitos B/inmunología , Linaje de la Célula , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas , Ratones , Modelos Inmunológicos , Receptores Notch , Transducción de SeñalRESUMEN
We found that Msx2-interacting nuclear target protein (MINT) competed with the intracellular region of Notch for binding to a DNA binding protein RBP-J and suppressed the transactivation activity of Notch signaling. Although MINT null mutant mice were embryonic lethal, MINT-deficient splenic B cells differentiated about three times more efficiently into marginal zone B cells with a concomitant reduction of follicular B cells. MINT is expressed in a cell-specific manner: high in follicular B cells and low in marginal zone B cells. Since Notch signaling directs differentiation of marginal zone B lymphocytes and suppresses that of follicular B lymphocytes in mouse spleen, the results indicate that high levels of MINT negatively regulate Notch signaling and block differentiation of precursor B cells into marginal zone B cells. MINT may serve as a functional homolog of Drosophila Hairless.
Asunto(s)
Linfocitos B/citología , Linfocitos B/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Nucleares/metabolismo , Animales , Subgrupos de Linfocitos B/citología , Subgrupos de Linfocitos B/metabolismo , Moléculas de Adhesión Celular , Diferenciación Celular , Proteínas de Unión al ADN/metabolismo , Drosophila , Proteínas de Drosophila , Marcación de Gen , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas , Hígado/citología , Hígado/embriología , Ratones , Ratones Noqueados , Proteínas Nucleares/deficiencia , Proteínas Nucleares/genética , Unión Proteica , Proteínas de Unión al ARN , Receptores Notch , Transducción de Señal , Activación TranscripcionalRESUMEN
The transcription factor recombination signal binding protein-J (RBP-J) functions immediately downstream of the cell surface receptor Notch and mediates transcriptional activation by the intracellular domain of all four kinds of Notch receptors. To investigate the function of RBP-J, we introduced loxP sites on both sides of the RBP-J exons encoding its DNA binding domain. Mice bearing the loxP-flanked RBP-J alleles, RBP-J(f/f), were mated with Mx-Cre transgenic mice and deletional mutation of the RBP-J gene in adult mice was induced by injection of the IFN-alpha inducer poly(I)-poly(C). Here we show that inactivation of RBP-J in bone marrow resulted in a block of T cell development at the earliest stage and increase of B cell development in the thymus. Lymphoid progenitors deficient in RBP-J differentiate into B but not T cells when cultured in 2'-deoxyguanosine-treated fetal thymic lobes by hanging-drop fetal thymus organ culture. Competitive repopulation assay also revealed cell autonomous deficiency of T cell development from bone marrow of RBP-J knockout mouse. Myeloid and B lineage differentiation appears normal in the bone marrow of RBP-J-inactivated mice. These results suggest that RBP-J, probably by mediating Notch signaling, controls T versus B cell fate decision in lymphoid progenitors.
Asunto(s)
Linfocitos B/inmunología , Linfocitos B/metabolismo , Proteínas de Unión al ADN/genética , Proteínas Nucleares , Linfocitos T/inmunología , Linfocitos T/metabolismo , Animales , Linfocitos B/citología , Trasplante de Médula Ósea , Diferenciación Celular , Ensayo de Unidades Formadoras de Colonias , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/metabolismo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/inmunología , Células Madre Hematopoyéticas/metabolismo , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas , Técnicas In Vitro , Linfopoyesis , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Receptores Notch , Transducción de Señal , Linfocitos T/citologíaRESUMEN
RBP-J is a key mediator of Notch signaling that regulates cell fate determination in various lineages. To investigate the function of Notch-RBP-J in mature B cell differentiation, we generated mice that selectively lacked B cell RBP-J expression using conditional mutagenesis. Absence of RBP-J led to the loss of marginal zone B (MZB) cells with a concomitant increase in follicular B cells; in contrast, B1 cells in the peritoneal cavity were unaffected. Lack of RBP-J caused no defects in B cells maintenance, survival, plasma cell differentiation or activation. It is therefore likely that Notch-RBP-J signaling regulates the lineage commitment of mature B cells into follicular versus MZB cells. In addition, in mice with RBP-J-deficient B cells, had no obvious changes in immunoglobulin production in response to Ficoll, lipopolysaccharide or chicken gammaglobulin. In contrast, these mice exhibited increased mortality rates after blood-borne bacterial infection, which indicates that MZB cells play pivotal roles in the clearance of these bacteria.