RESUMO
Little is known about tuftelin expression in the developing embryo, previously it was thought to play a role in tooth enamel mineralization. In this study we show tuftelin's spatio-temporal expression in mineralizing and nonmineralizing tissues of the craniofacial complex in the developing mouse embryo. Embryos aged E10.5-E18.5 and newborns aged P3 were used in this study. Polymerase chain reaction (PCR), Real-time PCR, sequencing, and in-situ hybridization were used to detect and quantify messenger RNA (mRNA) expression in different developmental stages. We applied indirect immunohistochemistry and western-blot analyses to investigate protein expression. Two tuftelin mRNA transcripts and a single 64KDa protein were detected throughout embryonic development. Tuftelin was detected in tissues which develop from different embryonic origins; ectoderm, ectomesenchyme, and mesoderm. Tuftelin mRNA and protein were expressed already at E10.5, before the initiation of tooth formation and earlier than previously described. The expression pattern of tuftelin mRNA and protein exhibits dynamic spatio-temporal changes in various tissues. Tuftelin is expressed in neuronal tissues, thus fitting with its described correlation to nerve growth factor. A shift between cytoplasmatic and perinuclear/nuclear expression implies a possible role in regulation of transcription. Recent studies showed tuftelin is induced under hypoxic conditions in-vitro and in-vivo, through the hypoxia-inducible factor 1-α pathway. These results led to the hypothesis that tuftelin is involved in adaptation to hypoxic conditions. The fact that much of mammalian embryogenesis occurs at O 2 concentrations of 1-5%, raises the possibility that tuftelin expression throughout development is due to its role in the adaptive mechanisms in response to hypoxia.
Assuntos
Proteínas do Esmalte Dentário/metabolismo , Cabeça/embriologia , Camundongos/embriologia , Animais , Animais Recém-Nascidos , Proteínas do Esmalte Dentário/genética , Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Distribuição TecidualRESUMO
Following myonecrosis, muscle satellite cells proliferate, differentiate and fuse, creating new myofibers. The Runx1 transcription factor is not expressed in naïve developing muscle or in adult muscle tissue. However, it is highly expressed in muscles exposed to myopathic damage yet, the role of Runx1 in muscle regeneration is completely unknown. Our study of Runx1 function in the muscle's response to myonecrosis reveals that this transcription factor is activated and cooperates with the MyoD and AP-1/c-Jun transcription factors to drive the transcription program of muscle regeneration. Mice lacking dystrophin and muscle Runx1 (mdx-/Runx1f/f), exhibit impaired muscle regeneration leading to age-dependent muscle waste, gradual decrease in motor capabilities and a shortened lifespan. Runx1-deficient primary myoblasts are arrested at cell cycle G1 and consequently differentiate. Such premature differentiation disrupts the myoblasts' normal proliferation/differentiation balance, reduces the number and size of regenerating myofibers and impairs muscle regeneration. Our combined Runx1-dependent gene expression, ChIP-seq, ATAC-seq and histone H3K4me1/H3K27ac modification analyses revealed a subset of Runx1-regulated genes that are co-occupied by MyoD and c-Jun in mdx-/Runx1f/f muscle. The data provide unique insights into the transcriptional program driving muscle regeneration and implicate Runx1 as an important participant in the pathology of muscle wasting diseases.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/fisiologia , Músculo Esquelético/fisiologia , Mioblastos/fisiologia , Regeneração , Animais , Sequência de Bases , Sítios de Ligação , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Sequência Consenso , Feminino , Expressão Gênica , Regulação da Expressão Gênica , Genes jun , Masculino , Camundongos Endogâmicos mdx , Proteína MyoD/metabolismoRESUMO
A fundamental aspect of skeletal myogenesis involves extensive rounds of cell fusion, in which individual myoblasts are incorporated into growing muscle fibers. Here we demonstrate that N-WASp, a ubiquitous nucleation-promoting factor of branched microfilament arrays, is an essential contributor to skeletal muscle-cell fusion in developing mouse embryos. Analysis both in vivo and in primary satellite-cell cultures, shows that disruption of N-WASp function does not interfere with the program of skeletal myogenic differentiation, and does not affect myoblast motility, morphogenesis and attachment capacity. N-WASp-deficient myoblasts, however, fail to fuse. Furthermore, our analysis suggests that myoblast fusion requires N-WASp activity in both partners of a fusing myoblast pair. These findings reveal a specific role for N-WASp during mammalian myogenesis. WASp-family elements appear therefore to act as universal mediators of the myogenic cell-cell fusion mechanism underlying formation of functional muscle fibers, in both vertebrate and invertebrate species.
Assuntos
Actinas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Músculos/citologia , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo , Animais , Diferenciação Celular , Fusão Celular , Células Cultivadas , Cruzamentos Genéticos , Drosophila , Heterozigoto , Camundongos , Camundongos Endogâmicos ICR , Modelos Biológicos , Desenvolvimento Muscular , Músculos/embriologia , Fatores de TempoRESUMO
Regeneration of mineralized tissues affected by chronic diseases comprises a major scientific and clinical challenge. Periodontitis, one such prevalent disease, involves destruction of the tooth-supporting tissues, alveolar bone, periodontal-ligament and cementum, often leading to tooth loss. In 1997, it became clear that, in addition to their function in enamel formation, the hydrophobic ectodermal enamel matrix proteins (EMPs) play a role in the regeneration of these periodontal tissues. The epithelial EMPs are a heterogeneous mixture of polypeptides encoded by several genes. It was not clear, however, which of these many EMPs induces the regeneration and what mechanisms are involved. Here we show that a single recombinant human amelogenin protein (rHAM(+)), induced in vivo regeneration of all tooth-supporting tissues after creation of experimental periodontitis in a dog model. To further understand the regeneration process, amelogenin expression was detected in normal and regenerating cells of the alveolar bone (osteocytes, osteoblasts and osteoclasts), periodontal ligament, cementum and in bone marrow stromal cells. Amelogenin expression was highest in areas of high bone turnover and activity. Further studies showed that during the first 2 weeks after application, rHAM(+) induced, directly or indirectly, significant recruitment of mesenchymal progenitor cells, which later differentiated to form the regenerated periodontal tissues. The ability of a single protein to bring about regeneration of all periodontal tissues, in the correct spatio-temporal order, through recruitment of mesenchymal progenitor cells, could pave the way for development of new therapeutic devices for treatment of periodontal, bone and ligament diseases based on rHAM(+).
Assuntos
Amelogenina/farmacologia , Regeneração Óssea/efeitos dos fármacos , Doenças do Cão/fisiopatologia , Ligamento Periodontal/efeitos dos fármacos , Periodontite/veterinária , Processo Alveolar/metabolismo , Processo Alveolar/fisiopatologia , Amelogenina/genética , Amelogenina/metabolismo , Animais , Linhagem Celular , Cemento Dentário/efeitos dos fármacos , Cemento Dentário/metabolismo , Cemento Dentário/fisiopatologia , Modelos Animais de Doenças , Doenças do Cão/genética , Doenças do Cão/metabolismo , Cães , Feminino , Humanos , Imuno-Histoquímica , Hibridização In Situ , Ligamento Periodontal/metabolismo , Ligamento Periodontal/fisiopatologia , Periodontite/fisiopatologia , Ratos , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Regeneração/efeitos dos fármacos , SpodopteraRESUMO
The amelogenins comprise 90% of the developing extracellular enamel matrix proteins and play a major role in the biomineralization and structural organization of enamel. Amelogenins were also detected, in smaller amounts, in postnatal calcifying mesenchymal tissues, and in several nonmineralizing tissues including brain. Low molecular mass amelogenin isoforms were suggested to have signaling activity; to produce ectopically chondrogenic and osteogenic-like tissue and to affect mouse tooth germ differentiation in vitro. Recently, some amelogenin isoforms were found to bind to the cell surface receptors; LAMP-1, LAMP-2 and CD63, and subsequently localize to the perinuclear region of the cell. The recombinant amelogenin protein (rHAM(+)) alone brought about regeneration of the tooth supporting tissues: cementum, periodontal ligament and alveolar bone, in the dog model, through recruitment of progenitor cells and mesenchymal stem cells. We show that amelogenin is expressed in various tissues of the developing mouse embryonic cranio-facial complex such as brain, eye, ganglia, peripheral nerve trunks, cartilage and bone, and is already expressed at E10.5 in the brain and eye, long before the initiation of tooth formation. Amelogenin protein expression was detected in the tooth germ (dental lamina) already at E13.5, much earlier than previously reported (E19). Application of amelogenin (rHAM(+)) beads together with DiI, on E13.5 and E14.5 embryonic mandibular mesenchyme and on embryonic tooth germ, revealed recruitment of mesenchymal cells. The present results indicate that amelogenin has an important role in many tissues of the cranio-facial complex during mouse embryonic development and differentiation, and might be a multifunctional protein.
Assuntos
Amelogenina/genética , Proteínas da Matriz Extracelular/fisiologia , Dente/crescimento & desenvolvimento , Amelogênese Imperfeita/genética , Animais , Desenvolvimento Ósseo , Osso e Ossos/embriologia , Cartilagem/embriologia , Cartilagem/crescimento & desenvolvimento , Proteínas do Esmalte Dentário/fisiologia , Éxons , Gânglios/embriologia , Gânglios/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Camundongos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Dente/embriologiaRESUMO
Tuftelin was initially found in the developing and mature extracellular enamel. Here we describe our novel discovery of tuftelin cellular distribution (protein and mRNA) in six soft tissues. The expression levels of tuftelin mRNA were significantly higher in mouse kidney and testis, in which oxygen levels are hovering closely to hypoxia under normal conditions.
Assuntos
Proteínas do Esmalte Dentário/análise , Expressão Gênica , Rim/química , Testículo/química , Animais , Western Blotting , Química Encefálica , Hipóxia Celular , Proteínas do Esmalte Dentário/genética , Olho/química , Imuno-Histoquímica , Hibridização In Situ , Rim/fisiologia , Fígado/química , Pulmão/química , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Confocal , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Testículo/fisiologia , Regulação para CimaRESUMO
The amelogenin protein is considered as the major molecular marker of developing ectodermal enamel. Recent data suggest other roles for amelogenin beyond structural regulation of enamel mineral crystal growth. Here we describe our novel discovery of amelogenin expression in long bone cells, in cartilage cells, in cells of the epiphyseal growth plate, and in bone marrow stromal cells.
Assuntos
Amelogenina/análise , Células da Medula Óssea/química , Cartilagem/química , Fêmur/química , Lâmina de Crescimento/química , Células-Tronco Mesenquimais/química , Tíbia/química , Amelogenina/química , Amelogenina/genética , Sequência de Aminoácidos , Animais , Cartilagem/citologia , Células Cultivadas , Cães , Fêmur/citologia , Expressão Gênica , Lâmina de Crescimento/citologia , Imuno-Histoquímica , Hibridização In Situ , Masculino , Microscopia Confocal , Dados de Sequência Molecular , Osteoblastos/química , Osteoclastos/química , Osteócitos/química , RNA Mensageiro/análise , Ratos , Análise de Sequência de Proteína , Células Estromais/química , Tíbia/citologiaRESUMO
The amelogenins are secreted by the ameloblast cells of developing teeth; they constitute about 90% of the enamel matrix proteins and play an important role in enamel biomineralization. Recent evidence suggests that amelogenin may also be involved in the regeneration of the periodontal tissues and that different isoforms may have cell-signalling effects. During enamel development and mineralization, the amelogenins are lost from the tissue due to sequential degradation by specific proteases, making isolation of substantial purified quantities of full-length amelogenin challenging. The aim of the present study was to express and characterize a recombinant human amelogenin protein in the eukaryotic baculovirus system in quantities sufficient for structural and functional studies. Human cDNA coding for a 175 amino acid amelogenin protein was subcloned into the pFastBac HTb vector (Invitrogen), this system adds a hexa-histidine tag and an rTEV protease cleavage site to the amino terminus of the expressed protein, enabling effective one-step purification by Ni2+-NTA affinity chromatography. The recombinant protein was expressed in Spodoptera frugiperda (Sf9) insect cells and the yield of purified his-tagged human amelogenin (rHAM+) was up to 10 mg/L culture. Recombinant human amelogenin (rHAM+) was characterized by SDS-PAGE, Western blot, ESI-TOF spectrometry, peptide mapping, and MS/MS sequencing. Production of significant amounts of pure, full-length amelogenin opened up the possibility to investigate novel functions of amelogenin. Our recent in vivo regeneration studies reveal that the rHAM+ alone could bring about regeneration of the periodontal tissues; cementum, periodontal ligament, and bone.
Assuntos
Baculoviridae/metabolismo , Proteínas do Esmalte Dentário/biossíntese , Amelogenina , Animais , Linhagem Celular , Clonagem Molecular , Proteínas do Esmalte Dentário/química , Proteínas do Esmalte Dentário/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Etiquetas de Sequências Expressas , Humanos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Sensibilidade e Especificidade , Espectrometria de Massas por Ionização por Electrospray/métodos , Spodoptera/metabolismoRESUMO
The amelogenin protein is considered as the major molecular marker of developing and mineralizing ectodermal enamel. It regulates the shape, size, and direction of growth of the enamel mineral crystallite. Recent data suggest other roles for amelogenin beyond regulation of enamel mineral crystal growth. The present study describes our recent discovery of amelogenin expression in soft tissues: in brain and in cells of the hematopoietic system, such as macrophages, megakaryocytes and in some of the hematopoietic stem cells. Reverse transcription-polymerase chain reaction (RT-PCR) followed by cDNA sequencing revealed, in mouse brain, two amelogenin mRNA isoforms: the full-length amelogenin including exon 4, and the isoform lacking exon 4. Immunohistochemistry revealed amelogenin expression in brain glial cells. Mouse macrophages were found to express the full-length amelogenin sequence lacking exon 4. Confocal microscopy revealed colocalization of amelogenin and CD41 (a megakaryocyte marker), as well as amelogenin and CD34 (a hematopoietic stem cell marker) in some of the bone marrow cells. The expression of amelogenin, a major structural protein of the mineralizing extracellular enamel matrix, also in cells of non-mineralizing soft tissues, suggests that amelogenin is multifunctional. Several different potential functions of amelogenin are discussed.