RESUMEN
Sarcopenia is a common complication of chronic kidney disease (CKD) and has a detrimental effect on prognosis. Previous studies have explored the role of secondary calciprotein particles (CPP2) in determining the progression of complications and poor outcomes in patients with CKD. However, no study has demonstrated that CPP2 impairs skeletal myogenesis. Our study revealed that CPP2 exposure inhibits skeletal myogenesis by suppressing myotube formation and expression of skeletal muscle-specific myosin heavy chain and actin in human primary myoblasts. Moreover, CPP2 exposure altered the expression patterns of lineage-determinative transcription factors responsible for regulating myotube differentiation marker genes. This study first demonstrated that CPP2 interferes with myoblast differentiation and myotube formation in vitro.
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Diferenciación Celular , Desarrollo de Músculos , Mioblastos , Humanos , Mioblastos/metabolismo , Mioblastos/citología , Células Cultivadas , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/citología , Cadenas Pesadas de Miosina/metabolismo , Cadenas Pesadas de Miosina/genética , Regulación de la Expresión GénicaRESUMEN
Fibrocartilaginous entheses consist of tendons, unmineralized and mineralized fibrocartilage, and subchondral bone, each exhibiting varying stiffness. Here we examined the functional role of sclerostin, expressed in mature mineralized fibrochondrocytes. Following rapid mineralization of unmineralized fibrocartilage and concurrent replacement of epiphyseal hyaline cartilage by bone, unmineralized fibrocartilage reexpanded after a decline in alkaline phosphatase activity at the mineralization front. Sclerostin was co-expressed with osteocalcin at the base of mineralized fibrocartilage adjacent to subchondral bone. In Scx-deficient mice with less mechanical loading due to defects of the Achilles tendon, sclerostin+ fibrochondrocyte count significantly decreased in the defective enthesis where chondrocyte maturation was markedly impaired in both fibrocartilage and hyaline cartilage. Loss of the Sost gene, encoding sclerostin, elevated mineral density in mineralized zones of fibrocartilaginous entheses. Atomic force microscopy analysis revealed increased fibrocartilage stiffness. These lines of evidence suggest that sclerostin in mature mineralized fibrochondrocytes acts as a modulator for mechanical tissue integrity of fibrocartilaginous entheses.
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Background: Sequential changes in brainstem and spinal cord neurons after traumatic injury to peripheral nerves are related to neuropathic pain symptoms. Purpose: This study was conducted to elucidate the influence of nerve insult on stimulus-induced c-Fos expression and ERK phosphorylation by brainstem neurons. Methods: The brainstem trigeminal sensory nuclear complex (BTSNC) was examined for neuronal profiles immunolabeled with c-Fos and phosphorylated ERK (p-ERK) antibodies elicited by stimulation of the tongue with capsaicin after lingual or inferior alveolar nerve (IAN) injury. Results: Abundant neuronal profiles immunolabeled for c-Fos and p-ERK elicited by capsaicin were distributed in the spinal trigeminal nucleus caudalis (Vc) without nerve injury. The spinal trigeminal nucleus oralis (Vo) contained limited numbers of these neuronal profiles after stimulation of the tongue. A significant reduction of these neuronal profiles in the ipsilateral Vc was detected after lingual nerve injury. After IAN injury, an increased number of neuronal profiles immunolabeled for c-Fos elicited by capsaicin was noted, while that of p-ERK was left unchanged in the ipsilateral Vc. On the both sides of the Vo, an increased number of capsaicin-induced neuronal profiles immunolabeled for c-Fos and p-ERK was detected after lingual or IAN injury. Conclusion: Differential effects of lingual or IAN injury on stimulus-induced c-Fos expression and ERK phosphorylation by Vo and Vc neurons may be involved in the complex nature of symptoms of trigeminal neuralgia.
RESUMEN
Peripheral nerve injuries produce a variety of negative structural and functional changes in the central terminal sites of damaged axons, as well as the injured primary afferents. Such changes have been shown to be involved in the development of neuropathic pain, which includes abnormal pain sensations such as allodynia and hyperalgesia. Since the spinal dorsal horn is the first central site where signals from peripheral sensory nerves are transmitted and shows a variety of changes after peripheral nerve injury or chronic inflammation of peripheral tissues, it is one of the most important sites contributing to the mechanisms underlying the development of neuropathic pain. The functional disruption of inhibitory interneurons and glial activation in the spinal dorsal horn after peripheral nerve injury cause reorganization of neuronal circuits and changes in the excitability of second-order neurons. These events are involved in the development or maintenance of neuropathic pain. Here, we describe the interactions of primary afferents, interneurons, and glial cells that may cause reorganization of synaptic inputs to spinal dorsal horn neurons after peripheral nerve injury.
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Neuralgia , Traumatismos de los Nervios Periféricos , Humanos , Traumatismos de los Nervios Periféricos/complicaciones , Neuralgia/etiología , Células del Asta Posterior , Asta Dorsal de la Médula Espinal , HiperalgesiaRESUMEN
Pseudomonas aeruginosa (P. aeruginosa) is associated with periapical periodontitis. The lesions are characterized by a disorder in osteoblast metabolism. Quorum sensing molecular N-(3-oxododecanoyl)-homoserine lactone (AHL) is secreted by P. aeruginosa and governs the expression of numerous virulence factors. AHL can trigger intracellular calcium ([Ca2+]i) fluctuations in many host cells. However, it is unclear whether AHL can regulate osteoblast metabolism by affecting [Ca2+]i changes or its spatial correlation. We explored AHL-induced apoptosis and differentiation in pre-osteoblastic MC3T3-E1 cells and evaluated [Ca2+]i mobilization using several extraction methods. The spatial distribution pattern of [Ca2+]i among cells was investigated by Moran's I, an index of spatial autocorrelation. We found that 30 µM and 50 µM AHL triggered opposing osteoblast fates. At 50 µM, AHL inhibited osteoblast differentiation by promoting mitochondrial-dependent apoptosis and negatively regulating osteogenic marker genes, including Runx2, Osterix, bone sialoprotein (Bsp), and osteocalcin (OCN). In contrast, prolonged treatment with 30 µM AHL promoted osteoblast differentiation concomitantly with cell apoptosis. The elevation of [Ca2+]i levels in osteoblasts treated with 50 µM AHL was spatially autocorrelated, while no such phenomenon was observed in 30 µM AHL-treated osteoblasts. The blocking of cell-to-cell spatial autocorrelation in the osteoblasts provoked by 50 µM AHL significantly inhibited apoptosis and partially restored differentiation. Our observations suggest that AHL affects the fate of osteoblasts (apoptosis and differentiation) by affecting the spatial correlation of [Ca2+]i changes. Thus, AHL acts as a double-edged sword for osteoblast function.
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4-Butirolactona/análogos & derivados , Calcio/metabolismo , Diferenciación Celular/efectos de los fármacos , Homoserina/análogos & derivados , Osteoblastos/patología , Periodontitis/microbiología , Pseudomonas aeruginosa/patogenicidad , 4-Butirolactona/toxicidad , Animales , Línea Celular , Homoserina/toxicidad , Ratones , Percepción de QuorumRESUMEN
Outer membrane vesicles (OMVs) are nanosized particles derived from the outer membrane of gram-negative bacteria. Oral bacterium Porphyromonas gingivalis (Pg) is known to be a major pathogen of periodontitis that contributes to the progression of periodontal disease by releasing OMVs. The effect of Pg OMVs on systemic diseases is still unknown. To verify whether Pg OMVs affect the progress of diabetes mellitus, we analyzed the cargo proteins of vesicles and evaluated their effect on hepatic glucose metabolism. Here, we show that Pg OMVs were equipped with Pg-derived proteases gingipains and translocated to the liver in mice. In these mice, the hepatic glycogen synthesis in response to insulin was decreased, and thus high blood glucose levels were maintained. Pg OMVs also attenuated the insulin-induced Akt/glycogen synthase kinase-3 ß (GSK-3ß) signaling in a gingipain-dependent fashion in hepatic HepG2 cells. These results suggest that the delivery of gingipains mediated by Pg OMV elicits changes in glucose metabolisms in the liver and contributes to the progression of diabetes mellitus.
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Membrana Externa Bacteriana/metabolismo , Cisteína-Endopeptidasas Gingipaínas/genética , Periodontitis/genética , Porphyromonas gingivalis/genética , Animales , Membrana Externa Bacteriana/patología , Modelos Animales de Enfermedad , Cisteína-Endopeptidasas Gingipaínas/metabolismo , Glucógeno Sintasa Quinasa 3 beta/genética , Humanos , Resistencia a la Insulina/genética , Hígado/metabolismo , Hígado/microbiología , Ratones , Periodontitis/microbiología , Periodontitis/patología , Porphyromonas gingivalis/metabolismo , Porphyromonas gingivalis/patogenicidad , Proteínas Proto-Oncogénicas c-akt/genética , Transducción de Señal/genéticaRESUMEN
Retinoic acid (RA), an active metabolite of vitamin A, plays pivotal roles in a wide variety of biological processes, such as body patterning, organ development, and cell differentiation and proliferation. RA signaling is mediated by nuclear retinoic acid receptors, α, ß, and γ (RARα, RARß, and RARγ). RA is a well-known regulator of cartilage and skeleton formation and RARs are also essential for skeletal growth and hypertrophic chondrocyte-specific gene expression. These important roles of RA and RARs in chondrogenesis have been widely investigated using in vivo mouse models. However, few reports are available on the function of each subtype of RARs on in vitro chondrocyte differentiation. Here, we examined the effect of specific agonists of RARs on chondrogenic differentiation of ATDC5 and C3H10T1/2 cells. Subtype-specific RAR agonists as well as RA decreased the expressions of chondrogenic differentiation marker genes and inhibited chondrogenic differentiation, which was accompanied with morphological change to spindle-shaped cells. Among RAR agonists, RARα and RARγ agonists revealed a strong inhibitory effect on chondrogenic differentiation. RARα and RARγ agonists also hampered viability of ATDC5 cells. These observations suggested that RARα and RARγ are dominant receptors of RA signaling that negatively regulate chondrogenic differentiation.
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Diferenciación Celular/efectos de los fármacos , Condrocitos/fisiología , Receptores de Ácido Retinoico/agonistas , Vitamina A/farmacología , Vitamina A/fisiología , Animales , Desarrollo Óseo/efectos de los fármacos , Diferenciación Celular/genética , Células Cultivadas , Condrogénesis , Depresión Química , Expresión Génica , Ratones , Osteogénesis/efectos de los fármacos , Receptores de Ácido Retinoico/fisiología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
Ability to form cellular aggregations such as tumorspheres and spheroids have been used as a morphological marker of malignant cancer cells and in particular cancer stem cells (CSC). However, the common definition of the types of cellular aggregation formed by cancer cells has not been available. We examined morphologies of 67 cell lines cultured on three dimensional morphology enhancing NanoCulture Plates (NCP) and classified the types of cellular aggregates that form. Among the 67 cell lines, 49 cell lines formed spheres or spheroids, 8 cell lines formed grape-like aggregation (GLA), 8 cell lines formed other types of aggregation, and 3 cell lines formed monolayer sheets. Seven GLA-forming cell lines were derived from adenocarcinoma among the 8 lines. A neuroendocrine adenocarcinoma cell line PC-3 formed asymmetric GLA with ductal structures on the NCPs and rapidly growing asymmetric tumors that metastasized to lymph nodes in immunocompromised mice. In contrast, another adenocarcinoma cell line DU-145 formed spheroids in vitro and spheroid-like tumors in vivo that did not metastasize to lymph nodes until day 50 after transplantation. Culture in the 3D nanoenvironment and in a defined stem cell medium enabled the neuroendocrine adenocarcinoma cells to form slowly growing large organoids that expressed multiple stem cell markers, neuroendocrine markers, intercellular adhesion molecules, and oncogenes in vitro. In contrast, the more commonly used 2D serum-contained environment reduced intercellular adhesion and induced mesenchymal transition and promoted rapid growth of the cells. In addition, the 3D stemness nanoenvironment promoted secretion of HSP90 and EpCAM-exosomes, a marker of CSC phenotype, from the neuroendocrine organoids. These findings indicate that the NCP-based 3D environment enables cells to form stem cell tumoroids with multipotency and model more accurately the in vivo tumor status at the levels of morphology and gene expression.
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Exosomas/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Nanotecnología , Células Madre Neoplásicas/patología , Organoides/patología , Animales , Hipoxia de la Célula , Línea Celular Tumoral , Perfilación de la Expresión Génica , Humanos , Metástasis Linfática , Ratones , Células Madre Neoplásicas/metabolismo , Organoides/metabolismoRESUMEN
Serine/threonine protein phosphatase 2A (PP2A) regulates diverse physiological processes such as cell cycle, growth, apoptosis, and signal transduction. Previously, we demonstrated that silencing of the α-isoform of PP2A catalytic subunit (PP2A Cα) in osteoblasts accelerated osteoblast differentiation, whereas its overexpression suppressed differentiation. In this study, we examined the role of PP2A Cα in in vivo bone formation by generating transgenic mice (PP2A-Tg), in which the dominant negative form of PP2A Cα was specifically expressed in osteoblasts. PP2A-Tg mice exhibited an increase in body weight, cortical bone mineral density, and cortical bone thickness. Interestingly, they also displayed higher amounts of adipose tissue in the bone marrow of tibiae. The co-culture study showed that PP2A Cα-knockdown osteoblasts stimulated adipocyte differentiation from undifferentiated mesenchymal cells via upregulation of the adipocyte marker genes, such as peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα). These results indicated that the reduction of PP2A Cα levels in osteoblasts promoted bone formation in vivo. Additionally, PP2A Cα in osteoblasts was also potentially involved in controlling adipocyte differentiation through a paracrine mechanism.
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Adipocitos/citología , Adipocitos/metabolismo , Diferenciación Celular , Osteogénesis , Proteína Fosfatasa 2/metabolismo , Adipogénesis , Animales , Biomarcadores/metabolismo , Densidad Ósea , Línea Celular , Colágeno Tipo I/metabolismo , Hueso Cortical/anatomía & histología , Técnicas de Silenciamiento del Gen , Ratones Transgénicos , Osteoblastos/metabolismo , Regulación hacia ArribaRESUMEN
The nuclear retinoic acid receptors (RARs) play key roles in skeletal development and endochondral ossification. Previously, we showed that RARγ regulates chondrogenesis and that pharmacological activation of RARγ blocked heterotopic ossification (HO), pathology in which endochondral bone forms in soft tissues. Thus, we reasoned that pharmacological inhibition of RARγ should enhance endochondral ossification, leading to a potential therapeutic strategy for bone deficiencies. We created surgical bone defects in wild type and RARγ-null mice and monitored bone healing. Fibrous, cartilaginous, and osseous tissues formed in both groups by day 7, but more cartilaginous tissue formed in mutants within and around the defects compared to controls. Next, we implanted a mixture of Matrigel and rhBMP2 subdermally to induce ectopic endochondral ossification. Administration of RARγ antagonists significantly stimulated ectopic bone formation in wild type but not in RARγ-null mice. The antagonist-induced increases in bone formation were preceded by increases in cartilage formation and were accompanied by higher levels of phosphorylated Smad1/5/8 (pSmad1/5/8) compared to vehicle-treated control. Higher pSmad1/5/8 levels were also observed in cartilaginous tissues forming in healing bone defects in RARγ-null mice, and increases in pSmad1/5/8 levels and Id1-luc activity were observed in RARγ antagonist-treated chondrogenic cells in culture. Our data show that genetic or pharmacological interference with RARγ stimulates endochondral bone formation and does so at least in part by stimulating canonical BMP signaling. This pharmacologic strategy could represent a new tool to enhance endochondral bone formation in the setting of various orthopedic surgical interventions and other skeletal deficiencies. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1096-1105, 2017.
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Regeneración Ósea/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Receptores de Ácido Retinoico/antagonistas & inhibidores , Retinoides/farmacología , Animales , Huesos/metabolismo , Evaluación Preclínica de Medicamentos , Femenino , Proteína 1 Inhibidora de la Diferenciación , Ratones , Distribución Aleatoria , Receptores de Ácido Retinoico/genética , Proteínas Smad/metabolismo , Receptor de Ácido Retinoico gammaRESUMEN
Heterotopic ossification (HO) consists of ectopic cartilage and bone formation following severe trauma or invasive surgeries, and a genetic form of it characterizes patients with Fibrodysplasia Ossificans Progressiva (FOP). Recent mouse studies showed that HO was significantly inhibited by systemic treatment with a corticosteroid or the retinoic acid receptor γ agonist Palovarotene. Because these drugs act differently, the data raised intriguing questions including whether the drugs affected HO via similar means, whether a combination therapy would be more effective or whether the drugs may hamper each other's action. To tackle these questions, we used an effective HO mouse model involving subcutaneous implantation of Matrigel plus rhBMP2, and compared the effectiveness of prednisone, dexamathaosone, Palovarotene or combination of. Each corticosteroid and Palovarotene reduced bone formation at max doses, and a combination therapy elicited similar outcomes without obvious interference. While Palovarotene had effectively prevented the initial cartilaginous phase of HO, the steroids appeared to act more on the bony phase. In reporter assays, dexamethasone and Palovarotene induced transcriptional activity of their respective GRE or RARE constructs and did not interfere with each other's pathway. Interestingly, both drugs inhibited the activity of a reporter construct for the inflammatory mediator NF-κB, particularly in combination. In good agreement, immunohistochemical analyses showed that both drugs markedly reduced the number of mast cells and macrophages near and within the ectopic Matrigel mass and reduced also the number of progenitor cells. In sum, corticosteroids and Palovarotene appear to block HO via common and distinct mechanisms. Most importantly, they directly or indirectly inhibit the recruitment of immune and inflammatory cells present at the affected site, thus alleviating the effects of key HO instigators.
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Antiinflamatorios/uso terapéutico , Osificación Heterotópica/tratamiento farmacológico , Pirazoles/uso terapéutico , Retinoides/agonistas , Estilbenos/uso terapéutico , Animales , Antiinflamatorios/farmacología , Peso Corporal/efectos de los fármacos , Cartílago/efectos de los fármacos , Cartílago/patología , Movimiento Celular/efectos de los fármacos , Dexametasona/farmacología , Dexametasona/uso terapéutico , Modelos Animales de Enfermedad , Quimioterapia Combinada , Genes Reporteros , Macrófagos/efectos de los fármacos , Macrófagos/patología , Mastocitos/efectos de los fármacos , Mastocitos/patología , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Osificación Heterotópica/patología , Prednisona/farmacología , Prednisona/uso terapéutico , Pirazoles/farmacología , Estilbenos/farmacología , Transfección , Resultado del TratamientoRESUMEN
Fibrodysplasia ossificans progressiva (FOP), a rare and as yet untreatable genetic disorder of progressive extraskeletal ossification, is the most disabling form of heterotopic ossification (HO) in humans and causes skeletal deformities, movement impairment, and premature death. Most FOP patients carry an activating mutation in a bone morphogenetic protein (BMP) type I receptor gene, ACVR1(R206H) , that promotes ectopic chondrogenesis and osteogenesis and, in turn, HO. We showed previously that the retinoic acid receptor γ (RARγ) agonist palovarotene effectively inhibited HO in injury-induced and genetic mouse models of the disease. Here we report that the drug additionally prevents spontaneous HO, using a novel conditional-on knock-in mouse line carrying the human ACVR1(R206H) mutation for classic FOP. In addition, palovarotene restored long bone growth, maintained growth plate function, and protected growing mutant neonates when given to lactating mothers. Importantly, palovarotene maintained joint, limb, and body motion, providing clear evidence for its encompassing therapeutic potential as a treatment for FOP. © 2016 American Society for Bone and Mineral Research.
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Receptores de Activinas Tipo I/genética , Extremidades/crecimiento & desarrollo , Extremidades/fisiopatología , Mutación/genética , Miositis Osificante/genética , Osificación Heterotópica/tratamiento farmacológico , Osificación Heterotópica/fisiopatología , Pirazoles/uso terapéutico , Estilbenos/uso terapéutico , Animales , Huesos/anomalías , Huesos/patología , Proliferación Celular/efectos de los fármacos , Condrocitos/efectos de los fármacos , Condrocitos/patología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Placa de Crecimiento/efectos de los fármacos , Placa de Crecimiento/patología , Homeostasis , Humanos , Ratones Transgénicos , Movimiento , Osificación Heterotópica/patología , Osteogénesis , Pirazoles/farmacología , Estilbenos/farmacologíaRESUMEN
Retinoic acid signaling regulates several biological events, including myogenesis. We previously found that retinoic acid receptor γ (RARγ) agonist blocks heterotopic ossification, a pathological bone formation that mostly occurs in the skeletal muscle. Interestingly, RARγ agonist also weakened deterioration of muscle architecture adjacent to the heterotopic ossification lesion, suggesting that RARγ agonist may oppose skeletal muscle damage. To test this hypothesis, we generated a critical defect in the tibialis anterior muscle of 7-week-old mice with a cautery, treated them with RARγ agonist or vehicle corn oil, and examined the effects of RARγ agonist on muscle repair. The muscle defects were partially repaired with newly regenerating muscle cells, but also filled with adipose and fibrous scar tissue in both RARγ-treated and control groups. The fibrous or adipose area was smaller in RARγ agonist-treated mice than in the control. In addition, muscle repair was remarkably delayed in RARγ-null mice in both critical defect and cardiotoxin injury models. Furthermore, we found a rapid increase in retinoid signaling in lacerated muscle, as monitored by retinoid signaling reporter mice. Together, our results indicate that endogenous RARγ signaling is involved in muscle repair and that selective RARγ agonists may be beneficial to promote repair in various types of muscle injuries.
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Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Receptores de Ácido Retinoico/agonistas , Tretinoina/farmacología , Cicatrización de Heridas/efectos de los fármacos , Animales , Ratones , Receptores de Ácido Retinoico/metabolismo , Retinoides/metabolismo , Transducción de Señal/efectos de los fármacos , Receptor de Ácido Retinoico gammaRESUMEN
To study the cellular mechanism of the tendon repair process, we used a mouse Achilles tendon injury model to focus on the cells recruited to the injured site. The cells isolated from injured tendon 1 week after the surgery and uninjured tendons contained the connective tissue progenitor populations as determined by colony-forming capacity, cell surface markers, and multipotency. When the injured tendon-derived progenitor cells (inTPCs) were transplanted into injured Achilles tendons, they were not only integrated in the regenerating area expressing tenogenic phenotype but also trans-differentiated into chondrogenic cells in the degenerative lesion that underwent ectopic endochondral ossification. Surprisingly, the micromass culture of the inTPCs rapidly underwent chondrogenic differentiation even in the absence of exogenous bone morphogenetic proteins or TGFßs. The cells isolated from human ruptured tendon tissues also showed connective tissue progenitor properties and exhibited stronger chondrogenic ability than bone marrow stromal cells. The mouse inTPCs contained two subpopulations one positive and one negative for CD105, a coreceptor of the TGFß superfamily. The CD105-negative cells showed superior chondrogenic potential in vitro and induced larger chondroid degenerative lesions in mice as compared to the CD105-positive cells. These findings indicate that tendon progenitor cells are recruited to the injured site of tendons and have a strong chondrogenic potential and that the CD105-negative population of these cells would be the cause for chondroid degeneration in injured tendons. The newly identified cells recruited to the injured tendon may provide novel targets to develop therapeutic strategies to facilitate tendon repair.
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Diferenciación Celular/fisiología , Células Madre Mesenquimatosas/citología , Células Madre/citología , Tendones/citología , Animales , Células Cultivadas , Condrogénesis/fisiología , Endoglina , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Osteogénesis/fisiología , Tendones/metabolismoRESUMEN
We have recently constructed a web-based database of gene expression in the mouse whole embryo, EMBRYS (http://embrys.jp/embrys/html/MainMenu.html). To allow examination of gene expression patterns to the fullest extent possible, this database provides both photo images and annotation data. However, since embryos develop via an intricate process of morphogenesis, it would be of great value to track embryonic gene expression from a three dimensional perspective. In fact, several methods have been developed to achieve this goal, but highly laborious procedures and specific operational skills are generally required. We utilized a novel microscopic technique that enables the easy capture of rotational, 3D-like images of the whole embryo. In this method, a rotary head equipped with two mirrors that are designed to obtain an image tilted at 45 degrees to the microscope stage captures serial images at 2-degree intervals. By a simple operation, 180 images are automatically collected. These 2D images obtained at multiple angles are then used to reconstruct 3D-like images, termed AERO images. By means of this system, over 800 AERO images of 191 gene expression patterns were captured. These images can be easily rotated on the computer screen using the EMBRYS database so that researchers can view an entire embryo by a virtual viewing on a computer screen in an unbiased or non-predetermined manner. The advantages afforded by this approach make it especially useful for generating data viewed in public databases.
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Embrión de Mamíferos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Imagenología Tridimensional/métodos , Animales , Bases de Datos Factuales , Embrión de Mamíferos/anatomía & histología , Femenino , Imagenología Tridimensional/instrumentación , Internet , Ratones , Ratones Endogámicos ICR , EmbarazoRESUMEN
BACKGROUND: The mechanism of tooth development is a complex process regulated by numerous genes including transcription factors, growth factors, and other intra- and extracellular molecules. Especially, transcription factors play a central role in gene expression, regulating a wide spectrum of biological processes including organogenesis. Substantial evidence has been demonstrated by a number of studies using genetically engineered animal models. However, detailed molecular mechanisms of tooth development have not been completely elucidated, partially because numerous genes that play essential roles in tooth development remain unidentified. RESULTS: In this study, we conducted an expression-based screening using gene expression database and in situ hybridization assays. Based on the gene expression database "EMBRYS," 207 out of 1,520 genes were expressed in the maxillary and/or mandibular processes and thus were selected for further analysis by section in situ hybridization. Among these candidates, 28 genes were newly identified as potential factors associated with tooth development by in situ hybridization assays with frontal sections of embryonic day 13.5 and 14.5 mouse embryos. The expression patterns were also examined at embryonic day 16.5 and 18.5. CONCLUSIONS: These results will contribute to elucidating the mechanisms of tooth development and to improving the technology for regeneration of tooth.
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Diente Molar/embriología , Diente Molar/metabolismo , Animales , Femenino , Hibridación in Situ , Ratones , Embarazo , Factores de Transcripción/metabolismoRESUMEN
Whole-mount in situ hybridization (WISH) is a method to visualize gene expression within a whole organism. Although it is an essential approach in developmental biology to describe the gene expression patterns during embryogenesis, the simultaneous processing of numerous embryos in a large-scale experiment such as a screening assay has been difficult for an individual due to the limited capacity of the experimental setting. This chapter describes the optimal protocol for a WISH assay in a relatively large experimental scale, which allows for more efficient processing of embryos. The major improvement in the efficiency has been achieved by the several refinements such as the introduction of a quick method for probe synthesis, the use of mesh-buckets to facilitate handling samples, and the simplification of the conventional procedures. In combination with publicly accessible gene expression databases, WISH assays will be more favorable for a large-scale assay using mouse embryos.
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Embrión de Mamíferos/metabolismo , Perfilación de la Expresión Génica/métodos , Hibridación in Situ/métodos , Animales , Bases de Datos Genéticas , Técnicas de Cultivo de Embriones , Femenino , Ensayos Analíticos de Alto Rendimiento/instrumentación , Ensayos Analíticos de Alto Rendimiento/métodos , Hibridación in Situ/instrumentación , Ratones , Biología Molecular/métodos , Embarazo , Sondas ARNRESUMEN
We created a whole-mount in situ hybridization (WISH) database, termed EMBRYS, containing expression data of 1520 transcription factors and cofactors expressed in E9.5, E10.5, and E11.5 mouse embryos--a highly dynamic stage of skeletal myogenesis. This approach implicated 43 genes in regulation of embryonic myogenesis, including a transcriptional repressor, the zinc-finger protein RP58 (also known as Zfp238). Knockout and knockdown approaches confirmed an essential role for RP58 in skeletal myogenesis. Cell-based high-throughput transfection screening revealed that RP58 is a direct MyoD target. Microarray analysis identified two inhibitors of skeletal myogenesis, Id2 and Id3, as targets for RP58-mediated repression. Consistently, MyoD-dependent activation of the myogenic program is impaired in RP58 null fibroblasts and downregulation of Id2 and Id3 rescues MyoD's ability to promote myogenesis in these cells. Our combined, multi-system approach reveals a MyoD-activated regulatory loop relying on RP58-mediated repression of muscle regulatory factor (MRF) inhibitors.
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Redes Reguladoras de Genes , Desarrollo de Músculos , Músculo Esquelético/embriología , Proteínas Represoras/metabolismo , Animales , Técnicas de Silenciamiento del Gen , Técnicas de Inactivación de Genes , Proteína 2 Inhibidora de la Diferenciación/metabolismo , Proteínas Inhibidoras de la Diferenciación/metabolismo , Ratones , Factores Reguladores Miogénicos/genéticaRESUMEN
The Caenorhabditis elegans heterochronic gene lin-28 regulates developmental timing in the nematode trunk. We report the dynamic expression patterns of Lin-28 homologues in mouse and chick embryos. Whole mount in situ hybridization revealed specific and intriguing expression patterns of Lin-28 in the developing mouse and chick limb bud. Mouse Lin-28 expression was detected in both the forelimb and hindlimb at E9.5, but disappeared from the forelimb at E10.5, and finally from the forelimb and hindlimb at E11.5. Chicken Lin-28, which was first detected in the limb primordium at stage 15/16, was also downregulated as the stage proceeded. The amino acid sequences of mouse and chicken Lin-28 genes are highly conserved and the similar expression patterns of Lin-28 during limb development in mouse and chicken suggest that this heterochronic gene is also conserved during vertebrate limb development.