RESUMO
Mesoderm induction begins during gastrulation. Recent evidence from several vertebrate species indicates that mesoderm induction continues after gastrulation in neuromesodermal progenitors (NMPs) within the posteriormost embryonic structure, the tailbud. It is unclear to what extent the molecular mechanisms of mesoderm induction are conserved between gastrula and post-gastrula stages of development. Fibroblast growth factor (FGF) signaling is required for mesoderm induction during gastrulation through positive transcriptional regulation of the T-box transcription factor brachyury We find in zebrafish that FGF is continuously required for paraxial mesoderm (PM) induction in post-gastrula NMPs. FGF signaling represses the NMP markers brachyury (ntla) and sox2 through regulation of tbx16 and msgn1, thereby committing cells to a PM fate. FGF-mediated PM induction in NMPs functions in tight coordination with canonical Wnt signaling during the epithelial to mesenchymal transition (EMT) from NMP to mesodermal progenitor. Wnt signaling initiates EMT, whereas FGF signaling terminates this event. Our results indicate that germ layer induction in the zebrafish tailbud is not a simple continuation of gastrulation events.
Assuntos
Transição Epitelial-Mesenquimal , Fatores de Crescimento de Fibroblastos/metabolismo , Mesoderma/embriologia , Sistema Nervoso/citologia , Sistema Nervoso/embriologia , Células-Tronco/citologia , Cauda/embriologia , Via de Sinalização Wnt , Sequência de Aminoácidos , Animais , Gástrula/metabolismo , Imageamento Tridimensional , Mesoderma/citologia , Mesoderma/metabolismo , Células-Tronco/metabolismo , Proteínas com Domínio T , Vimentina/química , Vimentina/metabolismo , Xenopus laevis/embriologia , Peixe-Zebra/embriologia , Proteínas de Peixe-ZebraRESUMO
Vertebrate body axis formation depends on a population of bipotential neuromesodermal cells along the posterior wall of the tailbud that make a germ layer decision after gastrulation to form spinal cord and mesoderm. Despite exhibiting germ layer plasticity, these cells never give rise to midline tissues of the notochord, floor plate and dorsal endoderm, raising the question of whether midline tissues also arise from basal posterior progenitors after gastrulation. We show in zebrafish that local posterior signals specify germ layer fate in two basal tailbud midline progenitor populations. Wnt signaling induces notochord within a population of notochord/floor plate bipotential cells through negative transcriptional regulation of sox2. Notch signaling, required for hypochord induction during gastrulation, continues to act in the tailbud to specify hypochord from a notochord/hypochord bipotential cell population. Our results lend strong support to a continuous allocation model of midline tissue formation in zebrafish, and provide an embryological basis for zebrafish and mouse bifurcated notochord phenotypes as well as the rare human congenital split notochord syndrome. We demonstrate developmental equivalency between the tailbud progenitor cell populations. Midline progenitors can be transfated from notochord to somite fate after gastrulation by ectopic expression of msgn1, a master regulator of paraxial mesoderm fate, or if transplanted into the bipotential progenitors that normally give rise to somites. Our results indicate that the entire non-epidermal posterior body is derived from discrete, basal tailbud cell populations. These cells remain receptive to extracellular cues after gastrulation and continue to make basic germ layer decisions.
Assuntos
Células-Tronco/citologia , Cauda/citologia , Proteínas de Peixe-Zebra/metabolismo , Animais , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Transdução de Sinais , Células-Tronco/fisiologia , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
The opportunistic human pathogen, Pseudomonas aeruginosa, is a major cause of infections in chronic wounds, burns and the lungs of cystic fibrosis patients. The P. aeruginosa genome encodes at least three proteins exhibiting the characteristic three domain structure of autotransporters, but much remains to be understood about the functions of these three proteins and their role in pathogenicity. Autotransporters are the largest family of secreted proteins in Gram-negative bacteria, and those characterised are virulence factors. Here, we demonstrate that the PA0328 autotransporter is a cell-surface tethered, arginine-specific aminopeptidase, and have defined its active site by site directed mutagenesis. Hence, we have assigned PA0328 with the name AaaA, for arginine-specific autotransporter of P. aeruginosa. We show that AaaA provides a fitness advantage in environments where the sole source of nitrogen is peptides with an aminoterminal arginine, and that this could be important for establishing an infection, as the lack of AaaA led to attenuation in a mouse chronic wound infection which correlated with lower levels of the cytokines TNFα, IL-1α, KC and COX-2. Consequently AaaA is an important virulence factor playing a significant role in the successful establishment of P. aeruginosa infections.
Assuntos
Aminopeptidases/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Infecções por Pseudomonas/enzimologia , Pseudomonas aeruginosa/patogenicidade , Fatores de Virulência/metabolismo , Infecção dos Ferimentos/enzimologia , Aminopeptidases/genética , Animais , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Doença Crônica , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Humanos , Camundongos , Mutagênese Sítio-Dirigida , Peptídeos/metabolismo , Infecções por Pseudomonas/genética , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/genética , Fatores de Virulência/genética , Infecção dos Ferimentos/genética , Infecção dos Ferimentos/microbiologiaRESUMO
The interface between bone tissue and metal implants undergoes various types of mechanical loading, such as strain, compression, fluid pressure, and shear stress, from daily activities. Such mechanical perturbations create suboptimal environments at the host bone-implant junction, causing an accumulation of wear particles and debilitating osseous integration, potentially leading to implant failure. While many studies have focused on the effect of particles on macrophages or osteoprogenitor cells, differential and combined effects of mechanical perturbations and particles on such cell types have not been extensively studied. In this study, macrophages and osteoprogenitor cells were subjected to physiological and superphysiological mechanical stimuli in the presence and absence of Ti particles with the aim of simulating various microenvironments of the host bone-implant junction. Macrophages and osteoprogenitor cells were capable of engulfing Ti particles through actin remodeling and also exhibited changes in mRNA levels of proinflammatory cytokines under certain conditions. In osteoprogenitor cells, superphysiological strain increased proinflammatory gene expression; in macrophages, such mechanical perturbations did not affect gene expression. We confirmed that this phenomenon in osteoprogenitor cells occurred via activation of the ERK1/2 signaling pathway as a result of damage to the cytoplasmic membrane. Furthermore, AZD6244, a clinically relevant inhibitor of the ERK1/2 pathway, mitigated particle-induced inflammatory gene expression in osteoprogenitor cells and macrophages. This study provides evidence of more inflammatory responses under mechanical strains in osteoprogenitor cells than macrophages. Phagocytosis of particles and mechanical perturbation costimulate the ERK1/2 pathway, leading to expression of proinflammatory genes.
Assuntos
Inflamação/induzido quimicamente , Inflamação/genética , Macrófagos/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Próteses e Implantes , Titânio/toxicidade , Actinas/metabolismo , Animais , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/genética , Membrana Celular/metabolismo , Microambiente Celular/efeitos dos fármacos , Microambiente Celular/genética , Citocinas/genética , Citocinas/metabolismo , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Inflamação/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Osteoblastos/metabolismo , Fagocitose/efeitos dos fármacos , Fagocitose/genéticaRESUMO
Owing to striking features of numerous multinucleated cells and bone destruction, giant cell tumor (GCT) of bone, often called as osteoclastoma, has drawn major attractions from orthopaedic surgeons, pathologists, and radiologists. The name GCT or osteoclastoma gives a false impression of a tumor comprising of proliferating osteoclasts or osteoclast precursors. The underlying mechanisms for excessive osteoclastogenesis are intriguing and GCT has served as an exciting disease model representing a paradigm of osteoclastogenesis for bone biologists. The modern interpretation of GCT is predominantly osteoclastogenic stromal cell tumors of mesenchymal origin. A diverse array of inflammatory cytokines and chemokines disrupts osteoblastic differentiation and promotes the formation of excessive multi-nucleated osteoclastic cells. Pro-osteoclastogenic cytokines such as receptor activator of nuclear factor kappa-B ligand (RANKL), interleukin (IL)-6, and tumor necrosis factor (TNF) as well as monocyte-recruiting chemokines such as stromal cell-derived factor-1 (SDF-1) and monocyte chemoattractant protein (MCP)-1 participate in unfavorable osteoclastogenesis and bone destruction. This model represents a self-sufficient osteoclastogenic paracrine loop in a localized area. Consistent with this paradigm, a recombinant RANK-Fc protein and bisphosphonates are currently being tried for GCT treatment in addition to surgical excision and conventional topical adjuvant therapies.