RESUMO
Male and female reproductive tracts develop from anterior intermediate mesoderm with similar differentiation processes. The anterior intermediate mesoderm develops into the mesonephros, and the Wolffian duct initiates by epithelialization in the mesonephros. The Müllerian duct invaginates from the coelomic epithelium of the cranial mesonephros for ductal formation and is then regionalized into proximal to caudal female reproductive tracts. In this study, we focused on the epithelialization of the Wolffian duct, initiation of the Müllerian duct, and the regionalization step of the Müllerian ducts as a continuous process. By using intermediate mesodermal cells from mouse pluripotent stem cells, we identified that inhibition of SMAD2/3 signaling might be involved in the differentiation into mesenchymal cells, after which mesonephric cells might be then epithelialized during differentiation of the Wolffian duct. Aggregation of coelomic epithelial cells might be related to initiation of the Müllerian duct. Transcriptomic analysis predicted that consensus sequences of SMAD3/4 were enriched among highly expressed genes in the proximal Müllerian duct. SMAD2/3 signaling to regulate differentiation of the Wolffian duct was continuously activated in the proximal Müllerian duct and was involved in proximal and oviductal regionalization. Therefore, SMAD2/3 signaling may be finely tuned to regulate differentiation from initiation to regionalization steps.
Assuntos
Ductos Paramesonéfricos , Ductos Mesonéfricos , Camundongos , Animais , Masculino , Feminino , Ductos Mesonéfricos/fisiologia , Ductos Paramesonéfricos/fisiologia , Diferenciação Celular , Células Epiteliais , Transdução de SinaisRESUMO
Extracellular vesicles (EVs), which are small membrane vesicles secreted from cells into bodily fluids, are promising candidates as biomarkers for various diseases. We propose a simple, highly sensitive method for detecting EVs using a microchip. The limit of detection (LOD) for EVs was improved 29-fold by changing the microchannel structure of the microchip and by optimizing the EV detection protocols. The height of the microchannel was changed from 25 to 8 µm only at the detection region, and the time for EV capture was extended from 5 to 10 min. The LOD was 6.3 × 1010 particles/mL, which is lower than the concentration of EVs in the blood. The detection time was 19 min, and the volume of EV solution used was 2.0 µL. These results indicate that an efficient supply of EVs to the detection region is effective in improving the sensitivity of EV detection. The proposed EV detection method is expected to contribute to the establishment of EV-based cancer point-of-care testing.
RESUMO
Cell cultures can simplify assays of biological phenomena; therefore, cell culture systems have been established for many species, even invertebrates. However, there are few primary culture systems from marine invertebrates that can be maintained long term. The Japanese scallop, Patinopecten yessoensis, is a marine bivalve. Cell culture systems for the scallop have only been established for a few organ-derived cell types and for embryonic cells. We developed a primary culture system for cells from male and female scallop gonads, hepatopancreas, and adductor muscle by utilizing culture conditions closer to those in nature, with regard to temperature, osmolarity, and nutrition. Primary cultured female gonadal cells were maintained for more than 1 month and had potential for proliferation. Furthermore, a genetic transfection system was attempted using a scallop-derived promoter and a lipofection reagent. GFP-positive cells were detected in the attempt. These technical developments would promote our understanding of biochemical mechanisms in scallops as well as providing clues for establishment of immortalized molluscan cell lines.
RESUMO
Oviduct, uterus, and vagina are derived from Müllerian ducts. But only in the vagina, the epithelium differentiates into stratified layers. Organ-specific secreted factors derived from the stroma of a neonatal mouse induce epithelial differentiation in the female reproductive tracts. However, the effects of the components and mechanical property of extracellular matrix (ECM) on the regulation of gene expression in the mesenchymal cells of neonatal stroma and differentiation of epithelium in the female reproductive tracts have been overlooked. In the present study, we have developed a simple 3D neonatal vaginal model using clonal cell lines to study the effect of ECM's components and stiffness on the epithelial stratification. Transcriptome analysis was performed by DNA-microarray to identify the components of ECM involved in the differentiation of vaginal epithelial stratification. The knockdown experiment of the candidate genes relating to vaginal epithelial stratification was focused on fibromodulin (Fmod), a collagen cross-linking protein. FMOD was essential for the expression of Bmp4, which encodes secreted factors to induce the epithelial stratification of vaginal mesenchymal cells. Furthermore, stiffer ECM as a scaffold for epithelial cells is necessary for vaginal epithelial stratification. Therefore, the components and stiffness of ECM are both crucial for the epithelial stratification in the neonatal vagina.
Assuntos
Proteína Morfogenética Óssea 4/genética , Diferenciação Celular , Células Epiteliais/fisiologia , Fibromodulina/genética , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Mesenquimais/fisiologia , Vagina/embriologia , Animais , Proteína Morfogenética Óssea 4/metabolismo , Elasticidade , Epitélio/embriologia , Matriz Extracelular/metabolismo , Feminino , Fibromodulina/metabolismo , CamundongosRESUMO
Uterine smooth muscle cells differentiate from mesenchymal cells, and gap junctions connect the muscle cells in the myometrium. At the neonatal stage, a uterine smooth muscle layer is situated away from the epithelium when smooth muscle cells are grafted near the epithelium, suggesting that the epithelium plays an important role in differentiation, proliferation, and/or migration of smooth muscle cells. In this study, developmental mechanisms regulating the formation of the smooth muscle layers in the mouse uterus were analyzed using an in vitro culture model. Differentiation of smooth muscle cells occurs at a neonatal stage because ACTA2 gene expression was increased at the outer layer, and GJA1 was not expressed in cellular membranes of uterine smooth muscle cells by postnatal day 15. To analyze the effects of the epithelium on the differentiation of smooth muscle cells, a bulk uterine mesenchymal cell line was established from p53-/- mice at postnatal day 3 (P3US cells). Co-culture with Müllerian ductal epithelial cells (E1 cells) induced repulsive migration of ACTA2-positive cells among bulk P3US cells from E1 cells, but it had no effects on the migration of any of 100% ACTA2-positive or negative smooth muscle cell lines cloned from P3US cells. Thus, uterine epithelial cells indirectly affected the repulsive migration of smooth muscle cells via mesenchymal cells. Conditioned medium by E1 cells inhibited differentiation into smooth muscle cells of clonal cells established from P3US cells. Therefore, the uterine epithelium inhibits the differentiation of stem-like progenitor mesenchymal cells adjacent to the epithelium into smooth muscle cells.
Assuntos
Células Epiteliais/fisiologia , Células-Tronco Mesenquimais/fisiologia , Ductos Paramesonéfricos/citologia , Músculo Liso/crescimento & desenvolvimento , Útero/crescimento & desenvolvimento , Actinas/genética , Actinas/metabolismo , Animais , Anticorpos , Diferenciação Celular , Movimento Celular , Técnicas de Cocultura , Feminino , Regulação da Expressão Gênica , Antígeno Ki-67/genética , Antígeno Ki-67/metabolismo , Camundongos , Camundongos Knockout , Proteína Supressora de Tumor p53/genética , Vimentina/genética , Vimentina/metabolismo , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
The mammary gland develops from the placode at ectodermal invagination. The rudimentary parenchyma (mammary bud) develops mammary trees and alveolar structures, suggesting that the mammary bud consists of stem/progenitor cells. Here, we established a clonal stem cell line from a mammary bud of a p53 null female embryo at day 14.5. FP5-3-1 line was a homogeneous cell population with polygonal epithelial morphology and spontaneously became heterogeneous during passages. Recloning gave rise to four sublines; three sublines have basal epithelial property and one subline has luminal epithelial property. The former sublines generate functional mammary glands when injected into cleared fat pads and the latter subline does not. The cell lines also express many stemness-related genes. The clonal cell lines established in the present study are shown to be mammary stem cells and not tumorigenic. They provide useful models for normal and tumor biology of the mammary gland in vivo and in vitro.
Assuntos
Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/embriologia , Tecido Adiposo/citologia , Animais , Linhagem Celular , Células Cultivadas , Feminino , Expressão Gênica , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos , Células-Tronco/citologia , Células-Tronco/fisiologia , Proteína Supressora de Tumor p53/genéticaRESUMO
In female mice, proximal, middle and caudal Müllerian ducts (MDs) differentiate into oviduct, uterus and vagina, respectively. The fates of female reproductive tract epithelia are determined by the mesenchyme. However, the mesenchymal fate determination system is still unclear. It is reported that presence or absence of retinoic acid (RA) signaling in MD mesenchyme induced uterine or vaginal mesenchyme, respectively. To analyze determination of the borderline, RA signal switching factors were found to play critical roles. Expression of a RA metabolizing enzyme, CYP26A1, was high in the epithelium of caudal MD and urogenital sinus, indicating that the enzyme causes the absence of RA signaling in the region. mRNA expression of some transcription factors regulating Aldh1a2, RA synthesis enzyme expressed in MDs, in other tissues was detected in MDs. When the transcription factor genes were overexpressed in a uterine mesenchymal cell line, C/ebpδ overexpression stimulated Aldh1a2 expression. Furthermore, C/EBPδ protein was strongly expressed in the proximal and middle regions of the MDs and bound to the Aldh1a2 promoter in vivo. Since C/ebpδ mRNA expression was maintained at the same level in proximal, middle and caudal MDs, we hypothesize that a high frequency of mitosis induces a low level protein expression in MD mesenchyme. In fact, the mitotic activity was significantly high in caudal mesenchyme, and a mathematical model showed that a gradient of protein was induced by cell proliferation. Therefore, morphogenesis of MDs controls the fate of mesenchyme via RA degradation in urogenital sinus and a gradient of proteins involved in RA synthesis.
RESUMO
Stratification of the vaginal epithelium is regulated by stromal factors. To analyze the mechanisms of stratification in vitro, 3 dimensional (3D) co-culture models were established with clonal cell lines. In the models, stromal cells were embedded in collagen gel and epithelial cells were seeded on the gel. In the 3D co-culture, stromal SV-6c4a1b cells induced epithelial stratification but stromal MV-1e6g1a cells did not, suggesting that SV-6c4a1b cells secrete molecules to induce stratification. Microarray analyses of these stromal cell lines identified chordin-like 1 (Chrd1) and WNT1 inducible signaling pathway protein 2 (Wisp2) as candidate genes inducing stratification. Chrdl1 variant1 and variant2 mRNAs were expressed not only in stromal SV-6c4a1b and MV-1e6g1a cells but also in epithelial SV-4b6b cells. Wisp2-overexpressing MV-1e6g1a cells, secreting WISP2 as much as SV-6c4a1b cells, induced stratification of epithelial cells. In addition, Wisp2-knockdowned SV-6c4a1b cells were unable to induce epithelial stratification. These results suggest that WISP2 is one of the stromal factors inducing stratification of the mouse vaginal epithelium.
Assuntos
Vagina/anatomia & histologia , Vagina/crescimento & desenvolvimento , Animais , Células Clonais/citologia , Células Clonais/metabolismo , Técnicas de Cocultura , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Epitélio/anatomia & histologia , Epitélio/crescimento & desenvolvimento , Epitélio/metabolismo , Estradiol/farmacologia , Proteínas do Olho/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Variação Genética , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Proteínas do Tecido Nervoso/genética , Progesterona/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/genética , Células Estromais/citologia , Células Estromais/metabolismo , Vagina/metabolismoRESUMO
The mouse vagina consists of stratified squamous epithelium and stroma and is regulated by ovarian hormones. Vaginal epithelial cells do not stratify, but rather form a monolayer and show an inconsistent responsiveness to ovarian hormones when cultured on plastic dish or matrix. To address the discrepancy between in vivo and in vitro observations, three-dimensional (3D) co-culture models are developed with clonal vaginal epithelial and stromal cell lines; stromal cells are embedded in collagen gel and epithelial cells are seeded on the gel. In the 3D models, epithelial cells express Transformation related protein 63 (Trp63) and begin to stratify when they are co-cultured with two out of three stromal cell lines, but not with the other stromal cell line. Stroma may consist of various types of cells with distinct functions.
Assuntos
Vagina/anatomia & histologia , Vagina/crescimento & desenvolvimento , Animais , Membrana Basal/metabolismo , Linhagem Celular , Células Clonais/citologia , Células Clonais/metabolismo , Técnicas de Cocultura , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Epitélio/efeitos dos fármacos , Epitélio/crescimento & desenvolvimento , Epitélio/metabolismo , Estradiol/farmacologia , Feminino , Camundongos , Camundongos Knockout , Modelos Anatômicos , Fosfoproteínas/deficiência , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células Estromais/citologia , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Transativadores/deficiência , Transativadores/genética , Transativadores/metabolismo , Vagina/metabolismoRESUMO
Teeth develop through interactions between epithelial and mesenchymal tissues mediated by a signaling network comprised of growth factors and transcription factors. However, little is known about how epigenetic modifiers affect signaling pathways and thereby regulate tooth formation. We previously reported that the histone 3 lysine 9 (H3K9) methyltransferase (MTase) G9a is specifically enriched in the tooth mesenchyme during mouse development. In this study, we investigated the functions of G9a in tooth development using G9a conditional knockout (KO) mice. We used Sox9-Cre mice to delete G9a in the tooth mesenchyme because Sox9 is highly expressed in the mesenchyme derived from the cranial neural crest. Immunohistochemical analyses revealed that G9a expression was significantly decreased in the mesenchyme of Sox9-Cre;G9afl/fl (G9a cKO) mice compared with that in Sox9-Cre;G9a fl/+(control) mice. Protein levels of the G9a substrate H3K9me2 were also decreased in the tooth mesenchyme. G9a cKO mice showed smaller tooth germ after embryonic day (E) 16.5 and E17.5, but not at E15.5. The developing cusp tips, which were visible in control mice, were absent in G9a cKO mice at E17.5. At 3 weeks after birth, small first molars with smaller cusps and unseparated roots were formed. Organ culture of tooth germs derived from E15.5 cKO mouse embryos showed impaired tooth development, suggesting that tooth development per se is affected independently of skull development. BrdU labeling experiments revealed that the proliferation rates were decreased in the mesenchyme in G9a cKO mice at E17.5. In addition, the proliferation rates in the tooth inner enamel epithelium were also decreased. In situ hybridization revealed altered localization of genes associated with tooth development. In cKO mice, intensively localized expression of mRNAs encoding bone morphogenic protein (Bmp2 and Bmp4) was observed in the tooth mesenchyme at E17.5, similar to the expression patterns observed in control mice at E15.5. Localization of Shh and related signaling components, including Gli1, Ptch1, and Ptch2, in the tooth mesenchyme of cKO mice was generally similar to that at earlier stages in control mice. In addition, expression of Fgf3 and Fgf10 in the mesenchyme was decreased in G9a cKO mice at P0. Expression levels of Fgf9 and p21, both of which were expressed in the secondary enamel-knot, were also decreased. Thus, the expression of genes associated with tooth development was delayed in cKO mice. Our results suggest that H3K9MTase G9a regulates cell proliferation and timing of differentiation and that G9a expression in the tooth mesenchyme is required for proper tooth development.
Assuntos
Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Histona-Lisina N-Metiltransferase/metabolismo , Dente/crescimento & desenvolvimento , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Epitélio/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Histona-Lisina N-Metiltransferase/genética , Mesoderma/citologia , Camundongos Transgênicos , Odontogênese/fisiologia , Fatores de Transcrição/metabolismoRESUMO
Many molluscs perform sex reversal, and sex hormones may be involved in the process. In adult scallops, Patinopecten yessoensis, gonadotropin releasing hormone and 17ß-estradiol (E2) are involved in male sexual maturation, however, little is known about the effects of E2 and testosterone (T) on the gonadal differentiation in young scallops. In the present study, scallop gonadal development was analyzed to determine the sex reversal stage in Funka bay, and effects of E2 and T were examined. In Funka bay, almost all scallops were male at month 12. Scallops equipped with ambiguous gonads were 61.1% at month 16 and disappeared at month 18. Therefore, sex reversal in Funka bay occurs at around month 16. For establishment of organ culture systems for bivalves, Manila clam gonads were cultured in 15% L-15 medium diluted with HBSS containing 10% KSR on agarose gel at 10°C, and the gonads survived for 14 days. Scallop gonads were also able to be cultured in 30% L15 medium diluted with ASW containing 10% KSR on agarose gel for seven days. At mature stage, Foxl2 and Tesk were predominantly expressed in ovary and testis, respectively. When scallop gonads at sex reversal stage were organ-cultured, sex steroid treatment decreased Tesk expression in the majority of scallop gonads at sex reversal stage. However, no obvious change in Foxl2 and Tesk expression was detected in mature gonads in response to either E2 or T in culture, suggesting sex steroid treatment might affect gonadal development at sex reversal stage.
Assuntos
Estradiol/farmacologia , Gônadas/efeitos dos fármacos , Gônadas/fisiologia , Técnicas de Cultura de Órgãos/métodos , Pectinidae/fisiologia , Testosterona/farmacologia , Animais , Feminino , Masculino , Diferenciação Sexual/efeitos dos fármacosRESUMO
Atopic dermatitis (AD), a chronic inflammatory skin disease, manifests as intractable itch, but its underlying mechanisms are poorly understood. This study assessed the relationship between immunoglobulin G (IgG) and dorsal root ganglia (DRG) in NC/Nga mice, a model of AD that manifests AD-like symptoms including itch. Immunohistochemical analysis showed large amounts of IgG in DRG extracts of NC/Nga mice with AD-like dermatitis, with a large fraction of the IgG distributed in satellite glial cells of the DRG. Proteomic analysis showed that this IgG was reactive against tropomyosin of Dermatophagoides farinae. These findings indicate that the accumulation of anti-tropomyosin IgG in DRG of atopic NC/Nga mice may be associated with the pathogenesis of AD-like symptoms, including itch.
Assuntos
Proteínas de Artrópodes/imunologia , Dermatite Atópica/imunologia , Dermatophagoides farinae/imunologia , Gânglios Espinais/imunologia , Imunoglobulina G/imunologia , Tropomiosina/imunologia , Sequência de Aminoácidos , Animais , Antígenos de Dermatophagoides/imunologia , Western Blotting , Dermatite Atópica/metabolismo , Modelos Animais de Doenças , Gânglios Espinais/metabolismo , Humanos , Imunoglobulina G/metabolismo , Imuno-Histoquímica , Masculino , Camundongos , Neuroglia/imunologia , Neuroglia/metabolismo , Proteoma/imunologia , Proteoma/metabolismo , Proteômica/métodos , Pele/imunologia , Pele/metabolismo , Pele/patologiaRESUMO
Extracellular vesicles (EVs) have been shown to transport miRNA, mRNA and protein, suggesting that they are new communication mediators. Diffusible mesenchymal factors determine the fate of Mullerian epithelial cells into oviductal ciliated cells. In the present study, we investigated whether EVs mediate the communication in the epithelial-mesenchymal interaction during oviductal ciliogenesis. EVs were isolated from cells of oviductal mesenchymal cell line (S1 cells) and characterized by TEM and expression of exosomal marker CD81. CD81 protein was also detected in oviductal mesenchyme, suggesting that CD81-expressing exosomes may be secreted from oviductal mesenchyme, as well as S1 cells. ß-actin, Gapdh and Vimentin mRNAs and miRNAs were detected in the exosomes. mRNA in S1 cells was able to be transported into cells of Mullerian epithelial cell line (E1 cells) via the exosomes. The effects of exosomes derived from S1 cells on ciliogenesis of E1 cells were analyzed by in vitro models. Culture with exosomes increased the number of ciliated cells in E1 cells. These results suggest that exosomes derived from mesenchymal cells modulate the oviductal ciliogenesis and open new avenues for developmental study of EVs.
Assuntos
Células Epiteliais/citologia , Vesículas Extracelulares/fisiologia , Tubas Uterinas/citologia , Células-Tronco Mesenquimais/citologia , Actinas/genética , Animais , Cílios , Células Epiteliais/metabolismo , Exossomos/metabolismo , Feminino , Células-Tronco Mesenquimais/metabolismo , Camundongos Endogâmicos , RNA Mensageiro/metabolismo , Tetraspanina 28/metabolismo , VimentinaRESUMO
Extracellular vesicles (EVs) are promising novel cancer biomarkers. However, rapid and easy analysis of EVs is challenging because conventional detection methods require large sample volumes and long detection times. Microchip-based analytical systems have particularly attracted attention for development of point-of-care (POC) diagnostics. Previously, various biomarker detection methods on a portable power-free poly(dimethylsiloxane) (PDMS) microchip using laminar flow-assisted dendritic amplification have been developed. Recently, for easy functionalization, we proposed a microchannel inner surface-functionalized power-free PDMS microchip (SF-PF microchip) utilizing electron beam-induced graft polymerization. In this study, we apply the technique and prepare a novel SF-PF microchip. On the microchip, EVs were successfully detected. The required sample volume was 1.0 µL, and the total analysis time was 20 min. The microchip can contribute to EV-based POC cancer diagnosis.
RESUMO
An improved organ culture system allowed to observe morphogenesis of mouse lingual papillae and taste buds relatively for longer period, in which fetal tongues were analyzed for 6 d. Taste cells were defined as eosinophobic epithelial cells expressing CK8 and Sox2 within lingual epithelium. Addition of glycogen synthase kinase 3 beta inhibitor CHIR99021 induced many taste cells and buds in non-gustatory and gustatory stratified lingual epithelium. The present study clearly demonstrated induction of taste cells and buds ectopically and without innervation.
Assuntos
Feto/fisiologia , Tecido Nervoso/fisiologia , Técnicas de Cultura de Órgãos/métodos , Papilas Gustativas/embriologia , Língua/embriologia , Animais , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Células Cultivadas , Epitélio/efeitos dos fármacos , Epitélio/inervação , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Glicogênio Sintase Quinase 3 beta/metabolismo , Proteínas Hedgehog/metabolismo , Camundongos Endogâmicos ICR , Morfogênese/efeitos dos fármacos , Tecido Nervoso/efeitos dos fármacos , Fosfolipase C beta/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Transporte Proteico/efeitos dos fármacos , Paladar/efeitos dos fármacos , Papilas Gustativas/efeitos dos fármacos , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/metabolismoRESUMO
Spinal itch transmission has been reported to be mediated by at least two neuronal populations in spinal dorsal horn, neurons expressing brain-natriuretic peptide (BNP) receptor (Npra) and gastrin-releasing peptide (GRP) receptor (GRPR). Although Npra-expressing neurons were shown to be upstream of GRPR- expressing neurons in spinal itch transmission, the roles of BNP and GRP in the spinal neurotransmission of histamine-dependent and -independent itch remains unclear. Using in vivo electrophysiology and behavior analysis, this study examined the responses of chloroquine (histamine-independent pruritogen)-responsive and histamine-responsive dorsal horn neurons to spinal applications of BNP and GRP. Electrophysiologically, 9.5% of chloroquine-responsive neurons responded to BNP, 33.3% to GRP, and 4.8% to both, indicating that almost half of chloroquine-responsive neurons were unresponsive to both BNP and GRP. In contrast, histamine-responsive neurons did not respond to spinal BNP application, whereas 30% responded to spinal GRP application, indicating that 70% of histamine-responsive neurons were unresponsive to both BNP and GRP. Behavioral analyses showed differences in the time-course and frequency of scratching responses evoked by intrathecal BNP and GRP. These findings provide evidence that most BNP-Npra and GRP-GRPR signaling involve different pathways of spinal itch transmission, and that multiple neurotransmitters, in addition to BNP and GRP, are involved in spinal itch transmission. The electrophysiological results also suggest that spinal BNP contributes little to histaminergic itch directly.
Assuntos
Peptídeo Liberador de Gastrina/fisiologia , Peptídeo Natriurético Encefálico/fisiologia , Células do Corno Posterior/efeitos dos fármacos , Células do Corno Posterior/fisiologia , Prurido/fisiopatologia , Potenciais de Ação , Animais , Cloroquina/administração & dosagem , Peptídeo Liberador de Gastrina/administração & dosagem , Histamina/administração & dosagem , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Peptídeo Natriurético Encefálico/administração & dosagem , Prurido/induzido quimicamenteRESUMO
We previously reported that in rats, chronic exposure to stress inhibits the induction of the common receptor (T1R3) for sweet and umami tastes. Here, we investigated whether endogenous glucocorticoids (GCs) might be responsible for this inhibition. In addition, we used mouse taste-bud cells (TB cells) expressing T1R3 to examine the effect of exogenous GC on T1R3 induction. Both adrenal glands were removed from rats [adrenalectomized (ADX) rats] and T1R3 mRNA expression in fungiform papillae was examined by real-time RT-PCR. T1R3 mRNA expression was significantly reduced in the ADX rats (versus sham-ADX rats). The reduced mRNA expression was restored to the level seen in the sham-ADX rats by administration of dexamethasone (DEX) at the smallest dose tested (0.1ng/kg, i.p.). However, with larger doses of DEX (10 and 1000ng/kg, i.p.) there was no such restoration (i.e., the expression level did not differ from that seen in ADX rats). Expression of the mRNA for the GC receptor-α was detected in mouse TB cells by RT-PCR. Significantly reduced T1R3 mRNA expression, as measured by real-time RT-PCR, was observed in TB cells at 24h after application of DEX (0.1, 1.0, or 10µM). These results suggest that in rodents: (a) a low concentration of endogenous GC is necessary and sufficient for induction of T1R3 expression, and that higher concentrations may actually inhibit such induction, and (b) this inhibitory effect may be due, at least in part, to a direct action of GC on taste cells.
Assuntos
Glucocorticoides/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Papilas Gustativas/metabolismo , Adrenalectomia , Animais , Linhagem Celular , Dexametasona/administração & dosagem , Dexametasona/análogos & derivados , Relação Dose-Resposta a Droga , Glucocorticoides/administração & dosagem , Masculino , Camundongos , RNA Mensageiro/metabolismo , Ratos Wistar , Receptores Acoplados a Proteínas G/efeitos dos fármacos , Receptores Acoplados a Proteínas G/genética , Receptores de Glucocorticoides/agonistas , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Papilas Gustativas/efeitos dos fármacos , Fatores de Tempo , Transfecção , Regulação para CimaRESUMO
Our previous study has confirmed that the distribution of oligodendrocyte precursor cells (OPCs) is disturbed in the embryonic cerebral cortex of Plexin-A4 knockout mice, and that Sema6A is expressed in OPCs in the region. The present study examined whether Sema6A expressed in OPCs is involved in their own migration, and used a clonal FBD-102b line as OPCs model. In an in vitro migration assay, Sema6A knockdown repressed the migration of FBD-102b cells. Additionally, in co-culture, 3T3 cells ectopically expressing Plexin-A4 were segregated from 3T3 cells ectopically expressing Sema6A. When FBD-102b cells were seeded in a spot and exposed to a gradient of both Sema3A and Sema6A, dispersion of FBD-102b cells was suppressed, and Plexin-A4 knockdown in FBD-102b cells attenuated the suppressive effect of the Semaphorins. These results indicate that Sema6A expressed in OPCs is involved in their autonomous migration through ligand-receptor interaction with Plexin-A4 expressed in surrounding cells.
Assuntos
Células-Tronco Embrionárias/metabolismo , Oligodendroglia/metabolismo , Semaforinas/metabolismo , Animais , Linhagem Celular , Movimento Celular , Células Clonais , Técnicas de Cocultura , Técnicas de Silenciamento de Genes , Camundongos , Camundongos Mutantes , Células NIH 3T3 , Proteínas do Tecido Nervoso , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Semaforina-3A/genética , Semaforina-3A/metabolismo , Semaforinas/genéticaRESUMO
Molecular mechanisms regulating positions of oligodendrocyte precursor cells (OPCs) remain unclear in developing cerebral cortex. To explore the mechanisms, we investigated how Plexin-A4, receptor of Semaphorin in OPCs, is involved in the positioning. We found that Plexin-A4 knockout mice exhibited (1) an increased number of OPCs in both the upper- and middle-regions of the cortical plate, where both indirect- and direct-ligands of Plexin-A4, Sema3A and Sema6A, respectively, were continuously expressed, and (2) aberrant distributions of OPCs in both the intermediate zone and corpus callosum, where Plexin-A4 was richly expressed in wild-type mice. These results suggest that Plexin-A4 is involved in the precise positioning of OPCs in developing cerebral cortex.
Assuntos
Padronização Corporal/fisiologia , Córtex Cerebral/embriologia , Córtex Cerebral/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/metabolismo , Oligodendroglia/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/deficiência , Células-Tronco Neurais/citologia , Oligodendroglia/citologia , Receptores de Superfície Celular/deficiênciaRESUMO
OBJECTIVES: p51/p63 gene, one of the p53 families, is specifically expressed in tooth germ epithelial cells and is essential for tooth development. This study aims to elucidate roles of p51/p63 in ameloblastic cell differentiation. MATERIALS AND METHODS: We determined expression pattern of each of p51/p63 isoforms by reverse transcriptase-polymerase chain reaction (RT-PCR) and western blotting using emtg (epithelium of molar tooth germ)-1, -2, -3, -4, and -5 cell lines established from a mandibular molar tooth germ of p53-deficient mice and SF2 cells which differentiates into ameloblasts upon exposure to NT4. Furthermore, we investigated the function of p51/p63 in these cells by Tet system, which enables inducible expression and knock down of the target genes of interest by exposing cells to doxycycline. RESULTS: The expression of ΔNp51B/ΔNp63α, an isoform without transactivation domain, was detected at high level in immature cells, while the expression of TAp51/TAp63 isoforms, isoforms of with the transactivation domain, was detected at high level in mature cells. Moreover, induction of TAp51A/TAp63γ expression led to down-regulation of ΔNp51B/ΔNp63α expression and cell proliferation. Interestingly, this also led to up-regulation of ameloblastin expression, a differentiation marker of amelogenesis. CONCLUSIONS: The results suggested that p51/p63 might regulate the cell proliferation and differentiation of tooth germ epithelial cells.