RESUMEN

It is unclear which nicotinic acetylcholine receptor (nAChR) subtypes are involved in the nicotinic activation of cells in the subfornical organ (SFO). We investigated the nAChR subtype using molecular biological, electrophysiological, pharmacological and immunohistochemical techniques. The use of reverse transcription-polymerase chain reaction in rats demonstrated the presence of mRNAs for the alpha2, alpha3, alpha4, alpha6, alpha7, beta2 and beta4 subunits in the SFO. The characteristics and dose-dependency of nicotine-induced inward currents in many dissociated SFO neurons were similar to those induced by acetylcholine in the presence of atropine. The nicotine-induced currents were larger than those induced by cytisine in most responding cells, suggesting the predominance of the beta2- rather than the beta4-containing nAChR. NIC-induced currents were significantly inhibited by dihydro-beta-erythroidine (a relatively selective antagonist for alpha4-containing nAChRs, and a partial antagonist for alpha2 or alpha3) at 300 nM in all responding cells. Additionally, the currents were significantly inhibited by alpha-conotoxin MII at 10 nM (a selective antagonist for alpha3- and/or alpha6-containing nAChRs) in some but not all responding cells. Methyllycaconitine at 10 nM (a selective alpha7-nAChR antagonist) reduced the nicotine-induced current significantly, but to a lesser extent. Fluorescence-labeled alpha-bungarotoxin (a homomeric alpha7 subtype selective binding drug) binding and immunofluorescence for the alpha7 subunit showed that positive images almost overlapped with those immunopositive for an astrocyte marker. These results suggest that the alpha4beta2 subtype is the main functional receptor in SFO neurons while alpha2, alpha3, alpha6, and beta4 subunits have some effect, and homomeric the alpha7 subtype exists in SFO astrocytes.

Asunto(s)

Neuroglía/metabolismo , Neuronas/metabolismo , Receptores Nicotínicos/metabolismo , Órgano Subfornical/metabolismo , Animales , Bungarotoxinas/metabolismo , Bungarotoxinas/farmacología , Relación Dosis-Respuesta a Droga , Estimulación Eléctrica , Electrofisiología , Fluoresceína-5-Isotiocianato , Colorantes Fluorescentes , Proteína Ácida Fibrilar de la Glía/metabolismo , Masculino , Nicotina/farmacología , Agonistas Nicotínicos/farmacología , Antagonistas Nicotínicos/farmacología , Técnicas de Placa-Clamp , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Receptor Nicotínico de Acetilcolina alfa 7

RESUMEN

Dental epithelial progenitor cells differentiate into various cell types during development of tooth germs. To study this mechanism, we produced immortalized dental epithelial progenitor cells derived from the cervical-loop epithelium of a rat lower incisor. The expression patterns of cytokeratin 14, nerve growth factor receptor p75, amelogenin, Notch2, and alkaline phosphatase were examined by immunohistochemistry in both lower and higher cell densities. The patterns of each were compared in the dental epithelium of rat lower incisors. The results demonstrated that these cells could produce ameloblast lineage cells, stratum intermedium cells, stellate reticulum, and outer enamel epithelium. Furthermore, fibroblast growth factor 10 stimulated proliferation of dental progenitor cells and subsequently increased the number of cells expressing alkaline phosphatase. These results suggest that fibroblast growth factor 10 plays a role in coupling mitogenesis of the cervical-loop cells and the production of stratum intermedium cells in rat incisors.

Asunto(s)

Células Madre/citología , Germen Dentario/citología , Fosfatasa Alcalina/análisis , Ameloblastos/citología , Amelogenina , Animales , Recuento de Células , División Celular/efectos de los fármacos , Linaje de la Célula , Células Cultivadas , Esmalte Dental/citología , Proteínas del Esmalte Dental/análisis , Células Epiteliales/citología , Factor 10 de Crecimiento de Fibroblastos , Factores de Crecimiento de Fibroblastos/farmacología , Incisivo , Queratinas/análisis , Mitógenos/farmacología , Odontogénesis , Ratas , Ratas Sprague-Dawley , Receptor de Factor de Crecimiento Nervioso/análisis , Receptor Notch2 , Receptores de Superficie Celular/análisis , Cuello del Diente/embriología

RESUMEN

Cholinergic muscarinic inputs to subfornical organ (SFO) neurones in rats were studied using histochemical, molecular-biological and electrophysiological techniques. Neurones in the medial septum and the diagonal band (MS-DBB) were retrogradely labelled by a tracer wheat germ agglutinin-conjugated horseradish peroxidase-colloidal gold complex injected into the SFO. Some in the MS-DBB were double-labelled by choline acetyltransferase (ChAT) antibody. Many ChAT-immunoreactive fibres were observed in the SFO. M3 muscarinic receptor subtype-like immunoreactivity, detected using a polyclonal antiserum, was observed in the SFO. In slice preparations, muscarine induced inward currents in a dose-related manner. The inward currents were suppressed by the relatively M3 muscarinic receptor selective antagonist 4-diphenylacetoxy-N-methylpiredine methiodide. In the whole-cell current mode, muscarine depolarized the membrane with increased frequency of action potentials. Reverse transcriptase-polymerase chain reaction showed the presence of M2-M5 receptor mRNA in the SFO tissues. These results suggest that the SFO receives cholinergic muscarinic synaptic inputs from the MS-DBB. Acetylcholine postsynaptically activates and depolarizes neurones in the SFO partly through specific muscarinic receptors, including M3 receptor subtypes.

Asunto(s)

Neuronas/fisiología , Receptores Muscarínicos/metabolismo , Órgano Subfornical/citología , Órgano Subfornical/fisiología , Acetilcolina/metabolismo , Animales , Colina O-Acetiltransferasa/análisis , Oro Coloide , Inmunohistoquímica , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Muscarina/farmacología , Agonistas Muscarínicos/farmacología , Antagonistas Muscarínicos/farmacología , Vías Nerviosas , Técnicas de Placa-Clamp , Piperidinas/farmacología , Ratas , Receptores Muscarínicos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Aglutinina del Germen de Trigo-Peroxidasa de Rábano Silvestre Conjugada , Aglutininas del Germen de Trigo

RESUMEN

The Sonic hedgehog (Shh)-Gli signaling pathway regulates development of many organs, including teeth. We cloned a novel gene encoding a transcription factor that contains a zinc finger domain with highest homology to the Gli family of proteins (61-64% amino acid sequence identity) from incisor pulp. Consistent with this sequence conservation, gel mobility shift assays demonstrated that this new Gli homologous protein, GliH1, could bind previously characterized Gli DNA binding sites. Furthermore, transfection assays in dental pulp cells showed that whereas Gli1 induces a nearly 50-fold increase in activity of a luciferase reporter containing Gli DNA binding sites, coexpression of Gli1 with Gli3 and/or GliH1 results in inhibition of the Gli1-stimulated luciferase activity. In situ hybridization analysis of mouse embryos demonstrated that GliH1 expression is initiated later than the three Gli genes and has a more restricted expression pattern. GliH1 is first detected diffusely in the limb buds at 10.0 days post coitus and later is expressed in the branchial arches, craniofacial interface, ventral part of the tail, whisker follicles and hair, intervertebral discs, teeth, eyes and kidney. LacZ was inserted into the GliH1 allele in embryonic stem cells to produce mice lacking GliH1 function. While this produced indicator mice for GliH1-expression, analysis of mutant mice revealed no discernible phenotype or required function for GliH1. A search of the Celera Genomics and associated databases identified possible gene sequences encoding a zinc finger domain with approximately 90% homology to that of GliH1, indicating there is a family of GliH genes and raising the possibility of overlapping functions during development.

Asunto(s)

Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/fisiología , Proteínas Oncogénicas/química , Proteínas Oncogénicas/fisiología , Factores de Transcripción/química , Factores de Transcripción/fisiología , Alelos , Secuencia de Aminoácidos , Animales , Sitios de Unión , Northern Blotting , Clonación Molecular , ADN/metabolismo , ADN Complementario/metabolismo , Proteínas de Unión al ADN/biosíntesis , Bases de Datos como Asunto , Pulpa Dental/metabolismo , Marcación de Gen , Glutatión Transferasa/metabolismo , Proteínas Hedgehog , Hibridación in Situ , Luciferasas/metabolismo , Ratones , Modelos Genéticos , Datos de Secuencia Molecular , Filogenia , Regiones Promotoras Genéticas , Unión Proteica , Estructura Terciaria de Proteína , ARN/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Factores de Tiempo , Transactivadores/metabolismo , Transfección , Proteína con Dedos de Zinc GLI1 , Dedos de Zinc

RESUMEN

Taste organs in the frog have a distinctive cell type located exclusively in the basal portion. In the same fashion as type III cells in mammalian taste buds, these basal cells show immunoreactivity for serotonin antibody. Further, these cells are morphologically similar to epidermal Merkel cells. To determine the significance of these serotonergic basal cells, we examined the early development of taste organs during metamorphosis of the frog by focusing on the origin and possible roles of serotonergic basal cells. For convenience of description, five stages of development (metamorphic stage to climax stages A-D) are defined. In the metamorphic stage, a few noninnervated Merkel cells appear at the upper layer of the lingual epithelium. No neuronal elements are seen in the epithelium at this stage. At climax stages A-B, immature fungiform papillae become discernible in the dorsal surface of the tongue, where the Merkel cells are located. Merkel cells then move downward and extend their cytoplasmic processes toward the basal lamina. These cells are identified by their intense immunoreactivity for serotonin. During the later stages, many nerve fibers in the subepithelial connective tissue approach the epithelium containing Merkel cells. At climax stages C-D, Merkel cells extend cytoplasmic processes along the basal lamina toward the center of the newly forming fungiform papillae. The morphology of these Merkel cells exactly coincides with that of serotonergic basal cells in adult taste organs. Profuse exocytotic release of dense-cored granules of Merkel cells toward the nerve fibers through the basal lamina is frequently seen in these stages. The present study indicates that serotonergic basal cells are derived from intraepithelial Merkel cells, which act as target sites for growing nerves and may be responsible for the initiation of taste organ morphogenesis.

Asunto(s)

Células de Merkel/fisiología , Metamorfosis Biológica/fisiología , Lengua/citología , Lengua/crecimiento & desarrollo , Animales , Inmunohistoquímica , Células de Merkel/metabolismo , Células de Merkel/ultraestructura , Microscopía Electrónica , Microscopía Electrónica de Rastreo , Serotonina/metabolismo , Lengua/ultraestructura

RESUMEN

We have cloned and characterized a new member of the bone morphogenetic protein/transforming growth factor beta (BMP/TGFbeta) superfamily, growth differentiation factor 11 (Gdf11), from rat incisor pulp RNA by reverse transcription-polymerase chain reaction using degenerate primers. The mature carboxyl-terminal domain encoded by Gdf11 is most closely related to Gdf8, being 90% identical to the mouse gene. Northern blot analysis revealed Gdf11 is expressed in adult dental pulp and brain. In situ hybridization of sections and whole-mount embryos demonstrated Gdf11 is first strongly expressed in restricted domains at 8.5 days post coitus (dpc) when it is highest in the tail bud. At 10.5 dpc, it is expressed in the branchial arches, limb bud, tail bud and posterior dorsal neural tube. Later, it is expressed in terminally-differentiated odontoblasts, the nasal epithelium, retina and specific regions of the brain.

Asunto(s)

Proteínas Morfogenéticas Óseas/biosíntesis , Desarrollo Embrionario y Fetal/genética , Regulación del Desarrollo de la Expresión Génica , Animales , Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/fisiología , Factores de Diferenciación de Crecimiento , Ratones , Morfogénesis

RESUMEN

Mash1, a mammalian homologue of the Drosophila achaete-scute proneural gene complex, plays an essential role in differentiation of subsets of peripheral neurons. In this study, using RT-PCR and in situ RT-PCR, we investigated if Mash1 gene expression occurs in rat taste buds. Further, we examined dynamics of Mash1 expression in the process of degeneration and regeneration in denervated rat taste buds. In rat tongue epithelium, Mash1 gene expression is confined to circumvallate, foliate, and fungiform papilla epithelia that include taste buds. In taste buds, Mash1-expressing cells are round cells in the basal compartment. In contrast, the mature taste bud cells do not express the Mash1 gene. Denervation and regeneration experiments show that the expression of Mash1 requires gustatory innervation. We conclude that Mash1 is expressed in cells of the taste bud lineage, and that the expression of Mash1 in rat taste buds is dependent upon gustatory innervation.

Asunto(s)

Proteínas de Unión al ADN/genética , Expresión Génica , Papilas Gustativas/metabolismo , Lengua/inervación , Factores de Transcripción/genética , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Linaje de la Célula , Desnervación , Células Epiteliales/metabolismo , Células Epiteliales/patología , Femenino , Nervio Glosofaríngeo/fisiología , Nervio Glosofaríngeo/cirugía , Masculino , Regeneración Nerviosa/fisiología , Etiquetado in Situ Primed , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Papilas Gustativas/citología , Papilas Gustativas/patología , Lengua/citología , Lengua/metabolismo , Lengua/patología

RESUMEN

The transforming growth factor (TGF)-beta superfamily comprises more than 35 structurally related genes that have been implicated in embryonic induction and morphogenesis. Different superfamily members may have distinct regulatory roles in tooth development and maintenance. Degenerate primer sets derived from the highly conserved carboxy terminal region of the TGF-beta superfamily were used for reverse transcriptase polymerase with poly(A)+ RNA from the rat incisor pulp as a template. TGF-beta superfamily members expressed in the pulp with known potential to differentiate into odontoblasts and to form dentine were identified. Nucleotide-sequence analysis of the amplified cDNAs identified those encoding activin-betaB; bone morphogenic protein (BMP)-2, -4, -7 and -8; growth/differentiation factor (GDF)-1, -5 and -6; and glial cell line-derived neurotrophic factor. In addition, Northern blot analysis detected TGF-beta1 -beta2 and -beta3; activin-betaA; BMP-6 and GDF-7 mRNA transcripts in the pulp. Coordinated expression of TGF-beta superfamily members in pulp may be critical in tooth development and repair.

Asunto(s)

Activinas , Pulpa Dental/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Oligopéptidos , Factor de Crecimiento Transformador beta/análisis , Animales , Northern Blotting , Médula Ósea/metabolismo , Proteína Morfogenética Ósea 2 , Proteína Morfogenética Ósea 4 , Proteína Morfogenética Ósea 7 , Proteínas Morfogenéticas Óseas/análisis , Proteínas Morfogenéticas Óseas/genética , Diferenciación Celular/genética , Dentina/citología , Dentina/metabolismo , Regulación de la Expresión Génica , Factor 1 de Diferenciación de Crecimiento , Factor 5 de Diferenciación de Crecimiento , Factor 6 de Diferenciación de Crecimiento , Sustancias de Crecimiento/análisis , Sustancias de Crecimiento/genética , Incisivo , Factores de Crecimiento Nervioso , Proteínas del Tejido Nervioso/análisis , Proteínas del Tejido Nervioso/genética , Odontoblastos/citología , Odontoblastos/metabolismo , Odontogénesis/genética , Péptidos/análisis , Péptidos/genética , Ratas , Ratas Endogámicas LEC , Factor de Crecimiento Transformador beta/genética

RESUMEN

Transforming growth factor-beta (TGF-beta) superfamily members and their cell-surface receptors may play inductive and/or regulatory roles in tooth development and repair. It will be important to identify the complete set of TGF-beta superfamily receptors, to examine their temporal and spatial localization during tooth development, and to elucidate the cascade of molecular events of tooth formation induced by the TGF-beta superfamily. In this report, we have cloned the cDNAs encoding potential receptors for TGF-beta superfamily members in rat incisor pulp and bovine adult pulp which are regarded as embryonic and adult pulp, respectively. We analyzed poly (A)+ RNA from rat incisor pulp and bovine adult pulp by reverse-transcriptase/polymerase chain-reaction (RT-PCR), using a degenerate primers corresponding to the most conserved amino acid sequences in the intracellular serine/threonine kinase of type I or type II like kinase-1 (ALK-1), ALK-2, ALK-3 (bone morphogenetic protein receptor type IA, BMPR-IA), ALK-4 (B1), ALK-5, ALK-6 (BMPR-IB), and BMPR-II (BMP type II receptor) was found to be in dental pulp. Northern blot analysis further detected TGF-beta type II receptor (T beta R-II) mRNA transcript in addition to the above-identified receptors. These results provide the first evidence of multiple type I and type II receptors for TGF-beta s, activins, and BMPs expressed in embryonic and adult pulp, implicating diverse function in tooth development and pulp tissue repair.

Asunto(s)

Pulpa Dental/metabolismo , Regulación de la Expresión Génica , Receptores de Factores de Crecimiento Transformadores beta/genética , Factor de Crecimiento Transformador beta/genética , Receptores de Activinas , Activinas , Secuencia de Aminoácidos , Animales , Northern Blotting , Proteínas Morfogenéticas Óseas/genética , Bovinos , Clonación Molecular , Cartilla de ADN , ADN Complementario/genética , Pulpa Dental/anatomía & histología , Pulpa Dental/crecimiento & desarrollo , Pulpa Dental/fisiología , Regulación del Desarrollo de la Expresión Génica , Sustancias de Crecimiento/genética , Incisivo , Inhibinas/genética , Datos de Secuencia Molecular , Odontogénesis/genética , Poli A/genética , Reacción en Cadena de la Polimerasa , Proteínas Serina-Treonina Quinasas/genética , ARN/genética , ARN Mensajero/genética , Ratas , Ratas Wistar , Receptores de Factores de Crecimiento/genética , Receptores de Factores de Crecimiento Transformadores beta/análisis , Factores de Tiempo , Transcripción Genética , Factor de Crecimiento Transformador beta/análisis , Cicatrización de Heridas/genética

RESUMEN

Transforming growth factor (TGF)-beta superfamily members and their receptors play a part in the differentiation of pulp cells into odontoblasts during reparative dentinogenesis. Bovine primary pulp-cell culture has been used as an in vitro model for proliferation and differentiation of pulp cells into preodontoblasts. To explore the molecular cascade of odontoblast differentiation, Northern blot analyses and reverse transcriptase polymerase chain reaction were here used to investigate the expression patterns of the genes for TGF-beta superfamily members: TGF-beta 1, namely bone morphogenetic protein (BMP)-4, BMP-7, activin-beta A and activin-beta B, and their type I and type II receptors, namely activin receptor-like kinase (ALK)-2 (ActR-I), ALK-3 (BMPR-IA), ALK-4 (ActR-IB), ALK-5 (T beta R-I), BMPR-II and T beta R-II, during differentiation of pulp cells into preodontoblasts in bovine adult pulp-cell culture. TGF-beta 1 and BMP-4 mRNAs were expressed from day 14 when matrix formation increased. BMP-7 mRNA was expressed only on day 28 when osteocalcin appeared. ALK-2 mRNA was increased from the beginning of the culture. ALK-3 and ALK-5 mRNAs first decreased on day 14 and increased again on day 21. T beta R-II and BMPR-II mRNAs were almost constant. These results suggest that the differentiation of pulp cells into preodontoblasts may be regulated by changes in the temporally coordinated expression pattern of TGF-beta superfamily members and their receptors, including up-regulation of transcription of TGF-beta 1, BMP-4, BMP-7, ALK-2, ALK-3, and ALK-5.

Asunto(s)

Regulación de la Expresión Génica/fisiología , Odontoblastos/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/genética , Factor de Crecimiento Transformador beta/genética , Secuencia de Aminoácidos , Animales , Northern Blotting/métodos , Southern Blotting/métodos , Bovinos , Diferenciación Celular , Células Cultivadas , Pulpa Dental/citología , Pulpa Dental/metabolismo , Humanos , Incisivo , Ratones , Datos de Secuencia Molecular , Odontoblastos/citología , Reacción en Cadena de la Polimerasa/métodos , ARN/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Análisis de Secuencia de ADN/métodos , Factores de Tiempo , Factor de Crecimiento Transformador beta/metabolismo