RESUMO
BACKGROUND: Xenopus laevis is a widely used model organism in the fields of genetics and development, and more recently evolution. At present, the most widely used staging table for X. laevis is based primarily on external features and does not describe the corresponding skull development in detail. Here, we describe skull development in X. laevis, complete with labeled figures, for each relevant stage in the most widely used staging table. RESULTS: We find skull development in X. laevis is, for the most part, distinct at each of the previously established stages based on external anatomy. However, variation does exist in the timing of onset of ossification of certain bones in the skull, which results in a range of stages where a skull element first ossifies. The overall sequence of ossification is less variable than the timing of ossification onset. CONCLUSIONS: While events in skull development vary somewhat between specimens, and in comparison, to external events, this staging table is useful in showing both when bones first appear and for documenting the range of temporal variance in X. laevis skull development more accurately than previously done. Furthermore, when only skull data are available, the approximate stage of a specimen can now be determined.
Assuntos
Cabeça , Crânio , Animais , Cartilagem , Osteogênese , Crânio/anatomia & histologia , Xenopus laevisRESUMO
Sutures are fibrous joints that occur between bone elements in vertebrate skulls, where they play a variety of roles including facilitating skull growth and function. In addition, a variety of studies examining sutures from diverse perspectives in many taxa have enabled the determination of anatomical homologs. Surprisingly, one important aspect of sutures-histology-remains unknown in the key model organism of the chicken. To fill this gap in our knowledge, we generated histological sections of six different cranial sutures across a range of developmental stages in embryonic chicken. Despite having a skull that is largely co-ossified or fused as an adult, we found that the types, components, and ontogeny of sutures in chicken skulls are very similar to sutures in other vertebrates. We did, however, find a new transient stage in the ontogeny of sutures between endochondral bone elements, in which one element has ossified and one was still cartilaginous. Moreover, to better understand the morphogenetic events at the onset of suture formation, we compared the developmental histology of six sutures with that of the space between the two ossification centers of the frontal-a location expected to be void of suture structures. We found that the mesenchymal cells in sutures condense and form a middle vascular layer. This was not found to be the case in the space between the two ossifications of the frontal, where instead only osteoid occurs.
Assuntos
Galinhas , Suturas Cranianas , Animais , Osteogênese , Crânio , SuturasRESUMO
Myosin phosphatase target subunit 1 (MYPT1), together with catalytic subunit of type1 delta isoform (PP1cdelta) and a small 20-kDa regulatory unit (M20), form a heterotrimeric holoenzyme, myosin phosphatase (MP), which is responsible for regulating the extent of myosin light chain phosphorylation. Here we report the identification and characterization of a molecular interaction between Seven in absentia homolog 2 (SIAH2) and MYPT1 that resulted in the proteasomal degradation of the latter in mammalian cells, including neurons and glia. The interaction involved the substrate binding domain of SIAH2 (aa 116-324) and a central region of MYPT1 (aa 445-632) containing a degenerate consensus Siah-binding motif RLAYVAP (aa 493-499) evolutionally conserved from fish to humans. These findings suggest a novel mechanism whereby the ability of MP to modulate myosin light chain might be regulated by the degradation of its targeting subunit MYPT1 through the SIAH2-ubiquitin-proteasomal pathway. In this manner, the turnover of MYPT1 would serve to limit the duration and/or magnitude of MP activity required to achieve a desired physiological effect.
Assuntos
Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Proteínas Nucleares/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Sequência de Aminoácidos , Animais , Astrócitos/metabolismo , Sítios de Ligação/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Sequência Consenso/fisiologia , Inibidores de Cisteína Proteinase/farmacologia , Citoplasma/metabolismo , Expressão Gênica/genética , Humanos , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , Mutação/fisiologia , Fosfatase de Miosina-de-Cadeia-Leve/genética , Neurônios/metabolismo , Proteínas Nucleares/genética , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma , Ligação Proteica/fisiologia , Domínios e Motivos de Interação entre Proteínas/fisiologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Transfecção , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina-Proteína Ligases/genéticaRESUMO
Embryonic staging tables provide a standard of developmental stages that can be used by individual investigators and provide approximate time points for the study of developmental phenomena. Surprisingly, despite the presence of a plethora of studies on the chicken skull and its role as a model species in developmental research, a staging table of the development of the chicken skull remains lacking. A detailed photographic staging table of the osseous portion of the chicken skull is thus presented here based on cleared and stained HH stages spanning HH 35 (first appearance of skull ossification) to the final stage before hatching (HH 45). This table documents the development of most of the cranial elements in the skull from the start of ossification until the element takes its final shape. The table shows that the elements of the lower jaw and ventral side of the skull begin ossifying before the skull roof and that most elements take roughly 5 days to reach their final shape, whereas others take up to 9 days (e.g., the frontal). The obtained results lead to several hypotheses about chicken skull development and provide a timeframe for future studies on chicken skull development.
Assuntos
Galinhas , Crânio , Animais , Cabeça , MandíbulaRESUMO
RPTPsigma is a cell adhesion molecule-like receptor protein tyrosine phosphatase involved in nervous system development. Its avian orthologue, known as cPTPsigma or CRYPalpha, promotes intraretinal axon growth and controls the morphology of growth cones. The molecular mechanisms underlying the functions of cPTPsigma are still to be determined, since neither its physiological ligand(s) nor its substrates have been described. Nevertheless, a major class of ligand(s) is present in the retinal basal lamina and glial endfeet, the potent native growth substrate for retinal axons. We demonstrate here that cPTPsigma is a heparin-binding protein and that its basal lamina ligands include the heparan sulfate proteoglycans (HSPGs) agrin and collagen XVIII. These molecules interact with high affinity with cPTPsigma in vitro, and this binding is totally dependent upon their heparan sulfate chains. Using molecular modelling and site-directed mutagenesis, a binding site for heparin and heparan sulfate was identified in the first immunoglobulin-like domain of cPTPsigma. HSPGs are therefore a novel class of heterotypic ligand for cPTPsigma, suggesting that cPTPsigma signaling in axons and growth cones is directly responsive to matrix-associated cues.
Assuntos
Proteínas Aviárias , Proteoglicanas de Heparan Sulfato/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Agrina/genética , Agrina/metabolismo , Animais , Membrana Basal/metabolismo , Sítios de Ligação/fisiologia , Embrião de Galinha , Colágeno/genética , Colágeno/metabolismo , Colágeno Tipo XVIII , Endostatinas , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Heparina/metabolismo , Isoenzimas/metabolismo , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Neuroglia/metabolismo , Neuroglia/ultraestrutura , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína/fisiologia , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases Semelhantes a Receptores , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores , Retina/embriologia , Retina/metabolismo , Homologia de Sequência de AminoácidosAssuntos
Linhagem da Célula , Músculo Esquelético/citologia , Músculo Esquelético/embriologia , Células Satélites de Músculo Esquelético/citologia , Animais , Biomarcadores/análise , Diferenciação Celular , Músculo Esquelético/metabolismo , Regeneração/fisiologia , Células Satélites de Músculo Esquelético/metabolismo , Somitos/citologia , Somitos/metabolismoRESUMO
Chick PTPsigma (cPTPsigma), also known as CRYPalpha, is a receptor-like protein tyrosine phosphatase found on axons and growth cones. Putative ligands for cPTPsigma are distributed within basement membranes and on glial end feet of the retina, optic nerve, and optic tectum, suggesting that cPTPsigma signaling is occurring along the whole retinotectal pathway. We have shown previously that cPTPsigma plays a role in supporting the retinal phase of axon outgrowth. Here we have now addressed the role of cPTPsigma within retinal axons as they undergo growth and topographic targeting in the optic tectum. With the use of retroviruses, a secretable cPTPsigma ectodomain was ectopically expressed in ovo in the developing chick optic tectum, with the aim of directly disrupting the function of endogenous cPTPsigma. In ovo, the secreted ectodomains accumulated at tectal sites in which cPTPsigma ligands are also specifically found, suggesting that they are binding to these endogenous ligands. Anterograde labeling of retinal axons entering these optic tecta revealed abnormal axonal phenotypes. These included the premature stalling and arborization of fibers, excessive pretectal arbor formation, and diffuse termination zones. Most of the defects were rostral of the predicted termination zone, indicating that cPTPsigma function is necessary for sustaining the growth of retinal axons over the optic tectum and for directing axons to their correct sites of termination. This demonstrates that regulation of cPTPsigma signaling in retinal axons is required for their topographic mapping, the first evidence of this function for a receptor-like protein tyrosine phosphatase in the retinotectal projection.
Assuntos
Proteínas Aviárias , Axônios/enzimologia , Proteínas Tirosina Fosfatases/fisiologia , Retina/embriologia , Colículos Superiores/citologia , Colículos Superiores/embriologia , Vias Aferentes , Animais , Axônios/fisiologia , Axônios/ultraestrutura , Embrião de Galinha , Vetores Genéticos , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases Semelhantes a Receptores , Retina/citologia , Retina/enzimologia , Retroviridae/genéticaRESUMO
Increased sarcolemmal permeability has been implicated as a major pathological event in muscular dystrophies. In our study, we evaluated whether damaged muscle fibres can be specifically targeted using albumin as a carrier. We tagged human serum albumin (HSA) with Gadolinium (Gd) and systemically applied this compound (Gd-DTPA-HSA) to wildtype and dystrophin-deficient mdx mice. We performed magnetic resonance imaging before and after intravenous administration of Gd-DTPA-HSA and found localised signal enhancement only in mdx skeletal muscle. We also examined skeletal muscle after contrast enhanced magnetic resonance imaging using anti-human albumin antibodies and demonstrated intracellular accumulation of Gd-DTPA-HSA in clusters of damaged mdx muscle fibres. Comparison of magnetic resonance imaging and histological data emphasised the value of contrast agent enhanced magnetic resonance imaging for the in vivo assessment of fibre damage in muscular dystrophies. Furthermore, our data provide evidence that albumin can be used as a carrier to target covalently bound molecules to degenerating muscle fibres.
Assuntos
Meios de Contraste/farmacocinética , Fibras Musculares Esqueléticas/patologia , Músculo Esquelético/patologia , Distrofia Muscular Animal/diagnóstico , Distrofia Muscular Animal/metabolismo , Albumina Sérica/farmacocinética , Animais , Anticorpos , Citoplasma/metabolismo , Modelos Animais de Doenças , Feminino , Gadolínio/farmacocinética , Imuno-Histoquímica , Imageamento por Ressonância Magnética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular Animal/fisiopatologia , Valor Preditivo dos Testes , Sarcolema/efeitos dos fármacos , Sarcolema/metabolismoRESUMO
This study determined the effect of decamethonium bromide (DMBr), a non-competitive blocker of the neuromuscular junction, on skeletal muscle development during chick embryogenesis. Decamethonium bromide caused generalized edema and high mortality with treated embryos rarely surviving beyond day 16 of incubation. Muscle degeneration was grossly evident on the muscles of abdomen, pectoral girdle, and leg. Semi-thin sections showed a high infiltration of macrophages in treated embryos and a massive degenerative process. Electron microscopy showed that both fast and slow fibers formed in the control and treated embryos, but those of the treated embryos failed to form myofibrils. Other organ systems, such as the heart and the gut, appeared histologically normal throughout the course of treatment. To investigate possible nerve independent action of DMBr on muscle development we determined the effect of this compound on the growth and differentiation of the C2C12 skeletal muscle cell line. DMBr treatment of C2C12 cell cultures did not affect the growth or survival of the cells, even at a tenfold higher concentration than that used in ovo, but myosin heavy chain expression was dramatically inhibited. We conclude that DMBr has a nerve independent blocking inhibition effect on myosin heavy chain synthesis in the developing avian embryo besides the recognized role as a non-competitive post-synaptic blocker of the neuromuscular junction.
Assuntos
Embrião de Galinha/efeitos dos fármacos , Embrião de Galinha/fisiologia , Compostos de Decametônio/farmacologia , Músculo Esquelético/embriologia , Fármacos Neuromusculares Despolarizantes/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Embrião de Galinha/metabolismo , Compostos de Decametônio/administração & dosagem , Relação Dose-Resposta a Droga , Músculo Esquelético/citologia , Músculo Liso/embriologia , Cadeias Pesadas de Miosina/antagonistas & inibidores , Fármacos Neuromusculares Despolarizantes/administração & dosagemRESUMO
Using whole-mount in situ hybridisation techniques, we have examined the expression of major components of the insulin-like growth factor (IGF) axis in early development of the chicken embryo, including both IGF-I and -II, the type 1 IGF receptor ( IGFR), and two of the IGF binding proteins, ( IGFBP) -2 and -5. We report that these genes fall into two distinct groups with respect to expression pattern, with IGFBP-2 displaying broad overlap of mRNA expression with IGFR and IGF-I during early development, whereas the expression profile of IGFBP-5 most closely resembled that of IGF-II. Comparison between different stages revealed IGFBP-2 mRNA was detected as early as stage 3, whereas IGFBP-5 was first seen at stage 4. In addition, we detected expression domains of IGFBP-5, and to a lesser extent IGFBP-2, which did not overlap with either IGFR or IGF expression patterns. This could indicate IGF independent actions of the IGFBPs during early embryonic development. A striking observation concerning the expression profiles of both IGF-II and IGFBP-5 at early stages of chick embryogenesis is that both these genes are expressed asymmetrically in a pattern similar to that of Sonic Hedgehog (Shh). Furthermore, using cyclopamine, we have demonstrated that IGFBP-5 expression in the early embryo is regulated by Shh. Taken together, these results describe an important role for the IGF system in the very early stages of the developing chicken embryo, and imply that IGFBP-2 and -5 are fundamental developmental factors, with the latter involved in Shh signalling pathways.
Assuntos
Padronização Corporal/genética , Embrião de Galinha/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Proteína 5 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Fator de Crescimento Insulin-Like I/genética , Transativadores/genética , Animais , Padronização Corporal/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Embrião de Galinha/citologia , Embrião de Galinha/metabolismo , DNA Complementar/análise , DNA Complementar/genética , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas Hedgehog , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/isolamento & purificação , Fator de Crescimento Insulin-Like II/genética , Dados de Sequência Molecular , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Receptor IGF Tipo 1/genética , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Alcaloides de Veratrum/farmacologiaRESUMO
In skeletal muscle, asymmetrically dividing satellite stem cells give rise to committed satellite cells that transcribe the myogenic determination factor Myf5, a Pax7-target gene. We identified the arginine methyltransferase Carm1 as a Pax7 interacting protein and found that Carm1 specifically methylates multiple arginines in the N terminus of Pax7. Methylated Pax7 directly binds the C-terminal cleavage forms of the trithorax proteins MLL1/2 resulting in the recruitment of the ASH2L:MLL1/2:WDR5:RBBP5 histone H3K4 methyltransferase complex to regulatory enhancers and the proximal promoter of Myf5. Finally, Carm1 is required for the induction of de novo Myf5 transcription following asymmetric satellite stem cell divisions. We defined the C-terminal MLL region as a reader domain for the recognition of arginine methylated proteins such as Pax7. Thus, arginine methylation of Pax7 by Carm1 functions as a molecular switch controlling the epigenetic induction of Myf5 during satellite stem cell asymmetric division and entry into the myogenic program.
Assuntos
Divisão Celular/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Fator de Transcrição PAX7/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Células Satélites de Músculo Esquelético/citologia , Transcrição Gênica , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Arginina/metabolismo , DNA/metabolismo , Células HEK293 , Histona-Lisina N-Metiltransferase , Histonas/metabolismo , Humanos , Lisina/metabolismo , Metilação , Camundongos , Dados de Sequência Molecular , Fator Regulador Miogênico 5/metabolismo , Fator de Transcrição PAX7/química , Fator de Transcrição PAX7/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Estrutura Terciária de Proteína , Células Satélites de Músculo Esquelético/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Especificidade por SubstratoRESUMO
BACKGROUND: MicroRNAs (miRNAs) are short non-coding RNAs predicted to regulate one third of protein coding genes via mRNA targeting. In conjunction with key transcription factors, such as the repressor REST (RE1 silencing transcription factor), miRNAs play crucial roles in neurogenesis, which requires a highly orchestrated program of gene expression to ensure the appropriate development and function of diverse neural cell types. Whilst previous studies have highlighted select groups of miRNAs during neural development, there remains a need for amenable models in which miRNA expression and function can be analyzed over the duration of neurogenesis. PRINCIPAL FINDINGS: We performed large-scale expression profiling of miRNAs in human NTera2/D1 (NT2) cells during retinoic acid (RA)-induced transition from progenitors to fully differentiated neural phenotypes. Our results revealed dynamic changes of miRNA patterns, resulting in distinct miRNA subsets that could be linked to specific neurodevelopmental stages. Moreover, the cell-type specific miRNA subsets were very similar in NT2-derived differentiated cells and human primary neurons and astrocytes. Further analysis identified miRNAs as putative regulators of REST, as well as candidate miRNAs targeted by REST. Finally, we confirmed the existence of two predicted miRNAs; pred-MIR191 and pred-MIR222 associated with SLAIN1 and FOXP2, respectively, and provided some evidence of their potential co-regulation. CONCLUSIONS: In the present study, we demonstrate that regulation of miRNAs occurs in precise patterns indicative of their roles in cell fate commitment, progenitor expansion and differentiation into neurons and glia. Furthermore, the similarity between our NT2 system and primary human cells suggests their roles in molecular pathways critical for human in vivo neurogenesis.
Assuntos
Perfilação da Expressão Gênica , MicroRNAs/genética , Sistema Nervoso/crescimento & desenvolvimento , Humanos , Análise de Sequência com Séries de OligonucleotídeosRESUMO
Skeletal muscle satellite cells play an essential role in muscle regeneration and exercise adaptation. In recent years atypical myogenic progenitors (non-satellite-cell muscle stem cells) have been identified in skeletal muscle and have been hypothesized to play an important role in the process of muscle regeneration. It remains unknown, however, whether any populations other than satellite cells play a significant role in repair and adaptation following exercise-induced damage. We assessed the response of the satellite cell population and the CD45+:Sca-1+ cell population, previously shown to support muscle regeneration following cardiotoxin-induced injury, after acute eccentrically biased exercise in wild-type mice. We observed evidence of focal muscle damage and repair following the exercise protocol using electron microscopy, hematoxylin-eosin staining, and single-fiber analysis. In addition, we observed an approximately sixfold increase in the number of Myf5-expressing cells by 48 h, which remained elevated until at least 96 h following exercise. We did not, however, observe any significant expansion of the CD45+:Sca-1+ cell population or commitment of resident CD45+:Sca-1+ cells to the myogenic lineage. Furthermore, expression of Wnt gene family members, previously associated with myogenic specification of CD45+:Sca-1+ cells, did not differ following exercise. Therefore, we conclude that muscle satellite cells are the primary responders to exercise-induced stress and that the CD45+:Sca-1+ myogenic progenitors do not contribute to muscle repair/adaptation following exercise.
Assuntos
Condicionamento Físico Animal/métodos , Células Satélites Perineuronais/fisiologia , Células Satélites de Músculo Esquelético/fisiologia , Animais , Antígenos Ly/metabolismo , Cardiotoxinas , Citometria de Fluxo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/fisiologia , Antígenos Comuns de Leucócito/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Microscopia Eletrônica de Varredura/métodos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Doenças Musculares/induzido quimicamente , Doenças Musculares/metabolismo , Doenças Musculares/patologia , Doenças Musculares/fisiopatologia , Fator Regulador Miogênico 5/genética , Células Satélites Perineuronais/metabolismo , Células Satélites Perineuronais/ultraestrutura , Células Satélites de Músculo Esquelético/metabolismo , Células Satélites de Músculo Esquelético/ultraestrutura , Fatores de Tempo , Proteínas Wnt/genética , Proteínas Wnt/metabolismoRESUMO
Satellite cells purified from adult skeletal muscle can participate extensively in muscle regeneration and can also re-populate the satellite cell pool, suggesting that they have direct therapeutic potential for treating degenerative muscle diseases. The paired-box transcription factor Pax7 is required for satellite cells to generate committed myogenic progenitors. In this study we undertook a multi-level approach to define the role of Pax7 in satellite cell function. Using comparative microarray analysis, we identified several novel and strongly regulated targets; in particular, we identified Myf5 as a gene whose expression was regulated by Pax7. Using siRNA, fluorescence-activated cell sorting (FACS) and chromatin immunoprecipitation (ChIP) studies we confirmed that Myf5 is directly regulated by Pax7 in myoblasts derived from satellite cells. Tandem affinity purification (TAP) and mass spectrometry were used to purify Pax7 together with its co-factors. This revealed that Pax7 associates with the Wdr5-Ash2L-MLL2 histone methyltransferase (HMT) complex that directs methylation of histone H3 lysine 4 (H3K4, refs 4-10). Binding of the Pax7-HMT complex to Myf5 resulted in H3K4 tri-methylation of surrounding chromatin. Thus, Pax7 induces chromatin modifications that stimulate transcriptional activation of target genes to regulate entry into the myogenic developmental programme.
Assuntos
Regulação da Expressão Gênica , Histona-Lisina N-Metiltransferase/metabolismo , Fator Regulador Miogênico 5/metabolismo , Fator de Transcrição PAX7/metabolismo , Animais , Linhagem Celular , Cromatina/metabolismo , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Eletroforese em Gel de Poliacrilamida , Citometria de Fluxo , Histona Metiltransferases , Histonas/metabolismo , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intracelular , Metilação , Camundongos , Camundongos Endogâmicos C57BL , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Mioblastos/citologia , Mioblastos/metabolismo , Fator Regulador Miogênico 5/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fator de Transcrição PAX7/genética , Ligação Proteica , Proteínas Metiltransferases , Proteínas/genética , Proteínas/metabolismo , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
We have previously shown that Myostatin, a member of the transforming growth factor beta (TFG-beta) family of signalling molecules, is expressed in developing muscle, and that treatment with recombinant Myostatin inhibited the expression of key myogenic transcription factors during chick embryogenesis. In this study, we followed the fate of muscle precursors after exposure to Myostatin. We report that in contrast to the down-regulation in expression of Pax-3, Myf-5, MyoD, and Myogenin, expression of Pax-7 was maintained. However, Myostatin completely inhibited cell division in the Pax-7-expressing cells. The inhibitory effect of Myostatin was reversible, as upon withdrawal myogenic cells re-initiated cell proliferation as well as expression of Pax-3 and MyoD. These results led us to investigate the temporal and spatial distribution of quiescent muscle precursors during development. To this end, we analysed distribution and mitotic behaviour of Pax-7-expressing cells during muscle development. Our studies revealed two populations of Pax-7-expressing cells, one that proliferated and incorporated BrdU, whilst the other did not. At early developmental stages, a high proportion of Pax-7-expressing cells proliferated, but there was a significant number of non-dividing Pax-7-expressing cells intermingled with differentiated muscle. Proliferating precursors became less frequent as development proceeded and at late fetal stages all Pax-7-expressing cells were mitotically quiescent. We suggest that Myostatin is an important signalling molecule responsible for imposing quiescence upon myogenic precursors during embryonic and foetal development.
Assuntos
Desenvolvimento Muscular , Músculo Esquelético/embriologia , Fatores de Regulação Miogênica/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Embrião de Galinha , Mitose , Desenvolvimento Muscular/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Proteína MyoD/metabolismo , Fator Regulador Miogênico 5/metabolismo , Miogenina/metabolismo , Miostatina , Fator de Transcrição PAX7/metabolismo , Fatores de Transcrição Box Pareados/metabolismo , Fator de Crescimento Transformador beta/farmacologiaRESUMO
The trefoil factor family (TFF) peptides are important in gastro-intestinal mucosal protection and repair. Their mechanism of action remains unclear and receptors are sought. We aimed to identify and characterise proteins binding to TFF2. A fusion protein of mouse TFF2 with alkaline phosphatase was generated and used to probe 2-D protein blots of mouse stomach. The resulting spots were analysed by MS. The protein identified was characterised by bioinformatics, rapid amplification of cDNA ends, in situ hybridisation (ISH) and immunohistochemistry (IHC). Functional assays were performed in gastrointestinal cell lines. A single major murine protein was identified and named blottin. It was previously unknown as a translated product. Blottin is also present in rat and human; the latter gene is also known as GDDR. The predicted full-length proteins are 184 amino acids long (20 kDa), reducing to 164 amino acids (18 kDa) after signal peptide cleavage. ISH of gastrointestinal tissues shows abundant blottin mRNA in gastric surface and foveolar epithelium. IHC shows cytoplasmic staining for blottin protein, and by immunoelectron microscopy in mucus granules and Golgi stacks. Previous work showed that blottin is down-regulated in gastric cancers. Blottin contains a BRICHOS domain, and has 56% similarity with gastrokine-1. Cultured HT-29 cells express blottin and show increased DNA synthesis with antiblottin antibody; however, this effect is reversed by the immunising peptide. We have identified and characterised a TFF2-binding protein produced by gastric epithelium. Blottin may play a role in gastrointestinal mucosal protection and modulate gut epithelial cell proliferation.
Assuntos
Proteínas de Transporte/metabolismo , Mucosa Gástrica/metabolismo , Peptídeos/metabolismo , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/isolamento & purificação , Linhagem Celular , Linhagem Celular Tumoral , Endonucleases/genética , Endonucleases/metabolismo , Células HT29 , Humanos , Imuno-Histoquímica/métodos , Hibridização In Situ/métodos , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica , Dados de Sequência Molecular , Proteínas Musculares/análise , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Hormônios Peptídicos , Peptídeos/análise , Peptídeos/genética , Ligação Proteica , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Estômago/ultraestrutura , Fator Trefoil-2RESUMO
A population of myogenic progenitors termed satellite cells undertakes postnatal development and repair of skeletal muscle. Studies have indicated that atypical myogenic precursors can also participate in muscle regeneration. The source of this regenerative capacity has been attributed to "adult stem cells" that represent poorly understood multipotent cell lineages, believed to reside in all adult tissue populations. Here we review the origin and location of muscle satellite cells and stem cells, as well as the mechanisms by which they may be specified. We discuss how the experimental models utilized raise important questions regarding the validity of extrapolating these findings.
Assuntos
Desenvolvimento Muscular , Músculo Esquelético/citologia , Regeneração , Células-Tronco/citologia , Células-Tronco/fisiologia , Animais , Modelos Biológicos , Músculo Esquelético/fisiologiaRESUMO
Skeletal muscle atrophy has extreme adverse consequences. Molecular mechanisms that mediate the process of atrophy are not well defined. Recent studies have focused on diverse molecular cascades that control the activation of ubiquitin ligases, indicating that the involvement of the ubiquitin proteasome may be common to a range of atrophic stimuli.
Assuntos
Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Atrofia Muscular/metabolismo , Atrofia Muscular/patologia , Animais , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitinas/metabolismoRESUMO
Feathers are formed following a series of reciprocal signals between the epithelium and the mesenchyme. Initially, the formation of a dense dermis leads to the induction of a placode in the overlying ectoderm. The ectoderm subsequently signals back to the dermis to promote cell division. Sonic Hedgehog (Shh) is a secreted protein expressed in the ectoderm that has previously been implicated in mitogenic and morphogenetic processes throughout feather bud development. We therefore interfered with Shh signaling during early feather bud development and observed a dramatic change in feather form and prominence. Surprisingly, outgrowth did occur and was manifest as irregular, fused, and ectopic feather domains at both molecular and morphological levels. Experiments with Di-I and BrdU indicated that this effect was at least in part caused by the dispersal of previously aggregated proliferating dermal cells. We propose that Shh maintains bud development by localizing the dermal feather progenitors.
Assuntos
Plumas/embriologia , Transativadores/metabolismo , Fator de Crescimento Transformador beta , Animais , Biomarcadores , Proteína Morfogenética Óssea 2 , Proteína Morfogenética Óssea 4 , Proteínas Morfogenéticas Ósseas/metabolismo , Bromodesoxiuridina/metabolismo , Carbocianinas/metabolismo , Divisão Celular/fisiologia , Embrião de Galinha , Plumas/anormalidades , Plumas/citologia , Proteínas Hedgehog , Técnicas In Vitro , Botões de Extremidades/metabolismo , Transdução de Sinais , Pele/citologia , Pele/efeitos dos fármacos , Pele/embriologia , Transativadores/efeitos dos fármacos , Alcaloides de Veratrum/farmacologiaRESUMO
In higher vertebrates, the paraxial mesoderm undergoes a mesenchymal to epithelial transformation to form segmentally organised structures called somites. Experiments have shown that signals originating from the ectoderm overlying the somites or from midline structures are required for the formation of the somites, but their identity has yet to be determined. Wnt6 is a good candidate as a somite epithelialisation factor from the ectoderm since it is expressed in this tissue. In this study, we show that injection of Wnt6-producing cells beneath the ectoderm at the level of the segmental plate or lateral to the segmental plate leads to the formation of numerous small epithelial somites. Ectopic expression of Wnt6 leads to sustained expression of markers associated with the epithelial somites and reduced or delayed expression of markers associated with mesenchymally organised somitic tissue. More importantly, we show that Wnt6-producing cells are able to rescue somite formation after ectoderm ablation. Furthermore, injection of Wnt6-producing cells following the isolation of the neural tube/notochord from the segmental plate was able to rescue somite formation at both the structural (epithelialisation) and molecular level, as determined by the expression of marker genes like Paraxis or Pax-3. We show that Wnts are indeed responsible for the epithelialisation of somites by applying Wnt antagonists, which result in the segmental plate being unable to form somites. These results show that Wnt6, the only known member of this family to be localised to the chick paraxial ectoderm, is able to regulate the development of epithelial somites and that cellular organisation is pivotal in the execution of the differentiation programmes. We propose a model in which the localisation of Wnt6 and its antagonists regulates the process of epithelialisation in the paraxial mesoderm.