RESUMO
Bioelectrical signaling, intercellular communication facilitated by membrane potential and electrochemical coupling, is emerging as a key regulator of animal development. Gap junction (GJ) channels can mediate bioelectric signaling by creating a fast, direct pathway between cells for the movement of ions and other small molecules. In vertebrates, GJ channels are formed by a highly conserved transmembrane protein family called the Connexins. The connexin gene family is large and complex, presenting a challenge in identifying the specific Connexins that create channels within developing and mature tissues. Using the embryonic zebrafish neuromuscular system as a model, we identify a connexin conserved across vertebrate lineages, gjd4, which encodes the Cx46.8 protein, that mediates bioelectric signaling required for appropriate slow muscle development and function. Through a combination of mutant analysis and in vivo imaging we show that gjd4/Cx46.8 creates GJ channels specifically in developing slow muscle cells. Using genetics, pharmacology, and calcium imaging we find that spinal cord generated neural activity is transmitted to developing slow muscle cells and synchronized activity spreads via gjd4/Cx46.8 GJ channels. Finally, we show that bioelectrical signal propagation within the developing neuromuscular system is required for appropriate myofiber organization, and that disruption leads to defects in behavior. Our work reveals the molecular basis for GJ communication among developing muscle cells and reveals how perturbations to bioelectric signaling in the neuromuscular system_may contribute to developmental myopathies. Moreover, this work underscores a critical motif of signal propagation between organ systems and highlights the pivotal role played by GJ communication in coordinating bioelectric signaling during development.
RESUMO
The developmental determination of primary motoneurons was investigated by transplanting identified motoneurons in embryonic zebrafish to new spinal cord positions. Some cells moved from the new positions in which they were placed back to their original positions, thus it was difficult to evaluate whether they were determined. Among cells that remained in their new positions, those transplanted about 1 hour before axogenesis developed axonal trajectories that were appropriate for their original soma positions, whereas those transplanted 2 to 3 hours before axogenesis developed morphologies appropriate for their new soma positions. These results suggest that motoneuronal identity is determined before axogenesis.
Assuntos
Embrião não Mamífero/fisiologia , Neurônios Motores/transplante , Medula Espinal/embriologia , Animais , Transporte Axonal , Axônios/fisiologia , Interneurônios/citologia , Interneurônios/fisiologia , Neurônios Motores/citologia , Neurônios Motores/fisiologia , Peixe-ZebraRESUMO
The human genome was analyzed for evidence that genes had been laterally transferred into the genome from prokaryotic organisms. Protein sequence comparisons of the proteomes of human, fruit fly, nematode worm, yeast, mustard weed, eukaryotic parasites, and all completed prokaryote genomes were performed, and all genes shared between human and each of the other groups of organisms were collected. About 40 genes were found to be exclusively shared by humans and bacteria and are candidate examples of horizontal transfer from bacteria to vertebrates. Gene loss combined with sample size effects and evolutionary rate variation provide an alternative, more biologically plausible explanation.
Assuntos
Evolução Biológica , Transferência Genética Horizontal , Genes Bacterianos , Genoma Humano , Animais , Arabidopsis/genética , Bactérias/genética , Caenorhabditis elegans/genética , Biologia Computacional , Bases de Dados Factuais , Drosophila melanogaster/genética , Genoma , Humanos , Invertebrados/genética , Parasitos/genética , Filogenia , Plantas/genética , Proteoma , Saccharomyces cerevisiae/genética , Vertebrados/genéticaRESUMO
To facilitate molecular genetic analysis of vertebrate development, haploid genetics was used to construct a recombination map for the zebrafish Danio (Brachydanio) rerio. The map consists of 401 random amplified polymorphic DNAs (RAPDs) and 13 simple sequence repeats spaced at an average interval of 5.8 centimorgans. Strategies that exploit the advantages of haploid genetics and RAPD markers were developed that quickly mapped lethal and visible mutations and that placed cloned genes on the map. This map is useful for the position-based cloning of mutant genes, the characterization of chromosome rearrangements, and the investigation of evolution in vertebrate genomes.
Assuntos
Mapeamento Cromossômico , Peixe-Zebra/genética , Animais , Clonagem Molecular , Feminino , Marcadores Genéticos , Genótipo , Masculino , Mutação , Fenótipo , Reação em Cadeia da Polimerase , Sequências Repetitivas de Ácido Nucleico , SoftwareRESUMO
The 2,160,837-base pair genome sequence of an isolate of Streptococcus pneumoniae, a Gram-positive pathogen that causes pneumonia, bacteremia, meningitis, and otitis media, contains 2236 predicted coding regions; of these, 1440 (64%) were assigned a biological role. Approximately 5% of the genome is composed of insertion sequences that may contribute to genome rearrangements through uptake of foreign DNA. Extracellular enzyme systems for the metabolism of polysaccharides and hexosamines provide a substantial source of carbon and nitrogen for S. pneumoniae and also damage host tissues and facilitate colonization. A motif identified within the signal peptide of proteins is potentially involved in targeting these proteins to the cell surface of low-guanine/cytosine (GC) Gram-positive species. Several surface-exposed proteins that may serve as potential vaccine candidates were identified. Comparative genome hybridization with DNA arrays revealed strain differences in S. pneumoniae that could contribute to differences in virulence and antigenicity.
Assuntos
Genoma Bacteriano , Análise de Sequência de DNA , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/patogenicidade , Antígenos de Bactérias , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Vacinas Bacterianas , Composição de Bases , Metabolismo dos Carboidratos , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Cromossomos Bacterianos/genética , Biologia Computacional , Elementos de DNA Transponíveis , DNA Bacteriano/química , DNA Bacteriano/genética , Duplicação Gênica , Genes Bacterianos , Hexosaminas/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Recombinação Genética , Sequências Repetitivas de Ácido Nucleico , Especificidade da Espécie , Streptococcus pneumoniae/imunologia , Streptococcus pneumoniae/metabolismo , Virulência , Óperon de RNArRESUMO
The 5.67-megabase genome of the plant pathogen Agrobacterium tumefaciens C58 consists of a circular chromosome, a linear chromosome, and two plasmids. Extensive orthology and nucleotide colinearity between the genomes of A. tumefaciens and the plant symbiont Sinorhizobium meliloti suggest a recent evolutionary divergence. Their similarities include metabolic, transport, and regulatory systems that promote survival in the highly competitive rhizosphere; differences are apparent in their genome structure and virulence gene complement. Availability of the A. tumefaciens sequence will facilitate investigations into the molecular basis of pathogenesis and the evolutionary divergence of pathogenic and symbiotic lifestyles.
Assuntos
Agrobacterium tumefaciens/genética , Genoma Bacteriano , Análise de Sequência de DNA , Agrobacterium tumefaciens/classificação , Agrobacterium tumefaciens/patogenicidade , Agrobacterium tumefaciens/fisiologia , Aderência Bacteriana/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Cromossomos Bacterianos/genética , Conjugação Genética , Replicação do DNA , Genes Bacterianos , Genes Reguladores , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Filogenia , Plantas/microbiologia , Plasmídeos , Replicon , Rhizobiaceae/genética , Rhizobiaceae/fisiologia , Sinorhizobium meliloti/genética , Sinorhizobium meliloti/fisiologia , Simbiose , Virulência/genéticaRESUMO
The 2,272,351-base pair genome of Neisseria meningitidis strain MC58 (serogroup B), a causative agent of meningitis and septicemia, contains 2158 predicted coding regions, 1158 (53.7%) of which were assigned a biological role. Three major islands of horizontal DNA transfer were identified; two of these contain genes encoding proteins involved in pathogenicity, and the third island contains coding sequences only for hypothetical proteins. Insights into the commensal and virulence behavior of N. meningitidis can be gleaned from the genome, in which sequences for structural proteins of the pilus are clustered and several coding regions unique to serogroup B capsular polysaccharide synthesis can be identified. Finally, N. meningitidis contains more genes that undergo phase variation than any pathogen studied to date, a mechanism that controls their expression and contributes to the evasion of the host immune system.
Assuntos
Genoma Bacteriano , Neisseria meningitidis/genética , Neisseria meningitidis/patogenicidade , Análise de Sequência de DNA , Variação Antigênica , Antígenos de Bactérias/imunologia , Bacteriemia/microbiologia , Cápsulas Bacterianas/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Elementos de DNA Transponíveis , Evolução Molecular , Fímbrias Bacterianas/genética , Humanos , Meningite Meningocócica/microbiologia , Infecções Meningocócicas/microbiologia , Dados de Sequência Molecular , Mutação , Neisseria meningitidis/classificação , Neisseria meningitidis/fisiologia , Fases de Leitura Aberta , Óperon , Filogenia , Recombinação Genética , Sorotipagem , Transformação Bacteriana , Virulência/genéticaRESUMO
The complete genome sequence of the radiation-resistant bacterium Deinococcus radiodurans R1 is composed of two chromosomes (2,648,638 and 412,348 base pairs), a megaplasmid (177,466 base pairs), and a small plasmid (45,704 base pairs), yielding a total genome of 3,284, 156 base pairs. Multiple components distributed on the chromosomes and megaplasmid that contribute to the ability of D. radiodurans to survive under conditions of starvation, oxidative stress, and high amounts of DNA damage were identified. Deinococcus radiodurans represents an organism in which all systems for DNA repair, DNA damage export, desiccation and starvation recovery, and genetic redundancy are present in one cell.
Assuntos
Genoma Bacteriano , Cocos Gram-Positivos/genética , Mapeamento Físico do Cromossomo , Análise de Sequência de DNA , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Catalase/genética , Cromossomos Bacterianos/genética , Dano ao DNA , Reparo do DNA/genética , DNA Bacteriano/genética , Metabolismo Energético , Genes Bacterianos , Cocos Gram-Positivos/química , Cocos Gram-Positivos/classificação , Cocos Gram-Positivos/efeitos da radiação , Dados de Sequência Molecular , Fases de Leitura Aberta , Estresse Oxidativo , Plasmídeos , Tolerância a Radiação , Sequências Repetitivas de Ácido Nucleico , Superóxido Dismutase/genética , Thermus/química , Thermus/genética , Raios UltravioletaRESUMO
The role of cellular interactions in determining the fates of two identified motoneurons in the embryonic zebrafish was investigated by transplanting individual motoneurons from labeled donor embryos to unlabeled hosts. The results suggest that although these cells normally adopt different fates, they form an equivalence group in which one fate is primary and the other is secondary. Both cells are able to adopt the primary fate. A cell that has adopted the secondary fate can be induced to switch to the primary fate by ablating the cell that has adopted the primary fate, even many hours after axogenesis. Although interactions between the two cells appear to regulate which cell adopts the secondary fate, these interactions seem to be independent of neuromuscular activity.
Assuntos
Comunicação Celular/fisiologia , Neurônios Motores/citologia , Peixe-Zebra/embriologia , Animais , Movimento Celular/fisiologia , Neurônios Motores/fisiologia , Junção Neuromuscular/fisiologiaRESUMO
Primary motoneurons in embryonic zebrafish innervate cell-specific muscles. During pathfinding, motoneuronal growth cones encounter three distinct regions: a common pathway, a choice point, and separate cell-specific pathways. To learn whether the order in which these regions are encountered influences pathway choice, we transplanted individual motoneurons to the choice point region. These cells selected their appropriate cell-specific pathways. Thus, the sequence in which pathway regions are encountered may not be important for accurate path-finding, and the cell-specific pathways may be delineated by distinct cues that individual growth cones recognize. Moreover, these cues are unlikely to be general ones, since primary sensory neurons transplanted to the same location do not extend growth cones along the motoneuronal pathways.
Assuntos
Neurônios Motores/citologia , Músculos/inervação , Peixe-Zebra/embriologia , Animais , Movimento Celular/fisiologia , Células Cultivadas , Neurônios Motores/fisiologia , Neurônios Motores/transplante , Músculos/embriologia , Neurônios Aferentes/citologia , Neurônios Aferentes/fisiologiaRESUMO
We examined the arrangement and development of identified neurons in zebrafish embryos homozygous for the mutation spt-1, which acts autonomously and specifically to alter the development of precursors of trunk segmented mesoderm, resulting in muscle-deficient myotomes. We found that the mutation alters the morphology, number, and arrangement of identified motoneurons. By transplanting identified motoneurons between wild-type and mutant embryos, we found that the effect of the mutation was nonautonomous. We suggest that the segmental arrangement and proper axonal development of motoneurons may result from interactions with segmented mesoderm.
Assuntos
Neurônios Motores/citologia , Mutação , Medula Espinal/embriologia , Peixe-Zebra/embriologia , Animais , Axônios/fisiologia , Contagem de Células , Interneurônios/citologia , Interneurônios/fisiologia , Mesoderma/fisiologia , Neurônios Motores/fisiologia , Músculos/embriologia , Músculos/inervação , Neurônios Aferentes/citologia , Neurônios Aferentes/fisiologia , Medula Espinal/citologia , Peixe-Zebra/genéticaRESUMO
Developing motoneurons in zebrafish embryos follow a stereotyped sequence of axonal outgrowth and accurately project their axons to cell-specific target muscles. During axonal pathfinding, an identified motoneuron pioneers the peripheral motor pathway. Growth cones of later motoneurons interact with the pioneer via contact, coupling, and axonal fasciculation. In spite of these interactions, ablation of the pioneer motoneuron does not affect the ability of other identified motoneurons to select the pathways that lead to appropriate target muscles. We conclude that interactions between these cells during pathfinding are not required for accurate pathway selection.
Assuntos
Axônios/fisiologia , Neurônios Motores/fisiologia , Animais , Axônios/ultraestrutura , Condutividade Elétrica , Embrião não Mamífero/fisiologia , Potenciais da Membrana , Neurônios Motores/ultraestrutura , Peixe-ZebraRESUMO
The determination and analysis of complete genome sequences has led to the suggestion that horizontal gene transfer may be much more extensive than previously appreciated. Many of these studies, however, rely on evidence that could be generated by forces other than gene transfer including selection, variable evolutionary rates, and biased sampling.
Assuntos
Evolução Molecular , Transferência Genética Horizontal , Genoma Arqueal , Genoma Bacteriano , Modelos GenéticosRESUMO
Cells within equivalence groups interact via lateral specification to determine cell fates during development in Caenorhabditis elegans and other invertebrates. Populations of cells within the developing zebrafish have features similar to those of invertebrate equivalence groups. In a simple example, two identified zebrafish motoneurons behave as an equivalence pair in which one cell adopts a primary fate and interactions between the cells assign the other cell to a secondary fate. A more complicated situation exists for two initially equivalent populations of zebrafish neural crest cells. We consider whether mechanisms similar to those involved in fate specification within invertebrate equivalence groups also function furing fate specification in vertebrates.
Assuntos
Sistema Nervoso/embriologia , Peixe-Zebra/embriologia , Animais , Caenorhabditis elegans/embriologia , Indução Embrionária , Neurônios Motores/citologia , Crista Neural/citologia , Crista Neural/embriologiaRESUMO
OBJECTIVE: To examine clinical correlates of juvenile-onset OCD across the lifespan. METHOD: Data collected at the intake interview from 257 consecutive participants with juvenile-onset OCD (20 children, 44 adolescents and 193 adults) in a naturalistic study of the clinical course of OCD were examined. Participants and parents of juvenile participants completed a structured diagnostic interview, rater-administered severity measures and self-report questionnaires. RESULTS: Children and adolescents (i.e. juveniles) shared similar features with the exception of age at onset and OCD symptom expression. Clinically meaningful differences between juvenile and adult participants were also found. Compared with adults, juveniles were more likely to be male, recall an earlier age at OCD onset and have different lifetime comorbidity patterns. CONCLUSION: Juvenile-onset OCD symptom expression is remarkably similar across the lifespan. However, findings also suggest clinically meaningful differences between juveniles and adults. Future work using a prospective design will improve our understanding of course patterns of juvenile-onset OCD.
Assuntos
Transtorno Obsessivo-Compulsivo/epidemiologia , Adolescente , Adulto , Idade de Início , Idoso , Transtorno do Deficit de Atenção com Hiperatividade/diagnóstico , Transtorno do Deficit de Atenção com Hiperatividade/epidemiologia , Transtorno do Deficit de Atenção com Hiperatividade/psicologia , Criança , Comorbidade , Estudos Transversais , Transtornos da Alimentação e da Ingestão de Alimentos/diagnóstico , Transtornos da Alimentação e da Ingestão de Alimentos/epidemiologia , Transtornos da Alimentação e da Ingestão de Alimentos/psicologia , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Transtornos do Humor/diagnóstico , Transtornos do Humor/epidemiologia , Transtornos do Humor/psicologia , Transtorno Obsessivo-Compulsivo/diagnóstico , Transtorno Obsessivo-Compulsivo/psicologia , Pais/psicologia , Escalas de Graduação Psiquiátrica/estatística & dados numéricos , Estudos Retrospectivos , Autorrevelação , Índice de Gravidade de Doença , Distribuição por Sexo , Transtornos Relacionados ao Uso de Substâncias/diagnóstico , Transtornos Relacionados ao Uso de Substâncias/epidemiologia , Transtornos Relacionados ao Uso de Substâncias/psicologia , Inquéritos e QuestionáriosRESUMO
Two zebrafish motoneurons, CaP and VaP, are initially developmentally equivalent; later, CaP innervates ventral muscle, whereas VaP dies. Current models suggest that vertebrate motoneuron death results from failure to compete for limited, target-derived trophic support. In contrast, we provide evidence that zebrafish ventral muscle can support both CaP and VaP survival. However, VaP's growth cone is prevented from extending into ventral muscle by CaP-dependent interactions with identified muscle fibers, the muscle pioneers; this interaction breaks the initial equivalence of CaP and VaP. Thus, the processes mediating VaP death are more complex than failure to compete for trophic support, and may be important for correct spatial patterning.
Assuntos
Apoptose , Neurônios Motores/fisiologia , Músculo Esquelético/inervação , Peixe-Zebra/embriologia , Animais , Comunicação Celular , Sobrevivência Celular , Corantes Fluorescentes/metabolismo , Cones de Crescimento/fisiologia , Substâncias de Crescimento/farmacologia , Modelos Biológicos , Neurônios Motores/citologia , Neurônios Motores/efeitos dos fármacos , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Peixe-Zebra/fisiologiaRESUMO
BACKGROUND: Normal gut function requires rhythmic and coordinated movements that are affected by developmental processes, physical and chemical stimuli, and many debilitating diseases. The imaging and characterization of gut motility, especially regarding periodic, propagative contractions driving material transport, are therefore critical goals. Previous image analysis approaches have successfully extracted properties related to the temporal frequency of motility modes, but robust measures of contraction magnitude, especially from in vivo image data, remain challenging to obtain. METHODS: We developed a new image analysis method based on image velocimetry and spectral analysis that reveals temporal characteristics such as frequency and wave propagation speed, while also providing quantitative measures of the amplitude of gut motion. KEY RESULTS: We validate this approach using several challenges to larval zebrafish, imaged with differential interference contrast microscopy. Both acetylcholine exposure and feeding increase frequency and amplitude of motility. Larvae lacking enteric nervous system gut innervation show the same average motility frequency, but reduced and less variable amplitude compared to wild types. CONCLUSIONS & INFERENCES: Our image analysis approach enables insights into gut dynamics in a wide variety of developmental and physiological contexts and can also be extended to analyze other types of cell movements.
Assuntos
Motilidade Gastrointestinal/fisiologia , Processamento de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Larva/fisiologia , Microscopia de Interferência/métodos , Reologia/métodos , Animais , Sistema Nervoso Entérico/fisiologia , Peixe-ZebraRESUMO
BACKGROUND: Fate mapping studies have shown that progenitor cells of three vertebrate embryonic midline structures - the floorplate in the ventral neural tube, the notochord and the dorsal endoderm - occupy a common region prior to gastrulation. This common region of origin raises the possibility that interactions between midline progenitor cells are important for their specification prior to germ layer formation. RESULTS: One of four known zebrafish homologues of the Drosophila melanogaster cell-cell signaling gene Delta, deltaA (dlA), is expressed in the developing midline, where progenitor cells of the ectodermal floorplate, mesodermal notochord and dorsal endoderm lie close together before they occupy different germ layers. We used a reverse genetic strategy to isolate a missense mutation of dlA, dlAdx2, which coordinately disrupts the development of floorplate, notochord and dorsal endoderm. The dlAdx2 mutant embryos had reduced numbers of floorplate and hypochord cells; these cells lie above and beneath the notochord, respectively. In addition, mutant embryos had excess notochord cells. Expression of a dominant-negative form of Delta protein driven by mRNA microinjection produced a similar effect. In contrast, overexpression of dlA had the opposite effect: fewer trunk notochord cells and excess floorplate and hypochord cells. CONCLUSION: Our results indicate that Delta signaling is important for the specification of midline cells. The results are most consistent with the hypothesis that developmentally equivalent midline progenitor cells require Delta-mediated signaling prior to germ layer formation in order to be specified as floorplate, notochord or hypochord.
Assuntos
Proteínas de Membrana/biossíntese , Peixe-Zebra/embriologia , Animais , Diferenciação Celular , Gástrula , Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana/genética , Mutação , NotocordaRESUMO
Genome sequence information has continued to accumulate at a spectacular pace during the past year. Details of the sequence and gene content of human chromosome 22 were published. The sequencing and annotation of the first two Arabidopsis thaliana chromosomes was completed. The sequence of chromosome 3 from Plasmodium falciparum, the second sequenced malaria chromosome, was reported, as was that of chromosome 1 from Leishmania major. The complete genomic sequences of five microbes were reported. Approaches to using data from completely sequenced microbial genomes in phylogenetic studies are being explored, as is the application of microarrays to whole genome expression analysis.
Assuntos
Genoma Humano , Genoma , Animais , Evolução Molecular , Humanos , FilogeniaRESUMO
All animals are ecosystems with resident microbial communities, referred to as microbiota, which play profound roles in host development, physiology, and evolution. Enabled by new DNA sequencing technologies, there is a burgeoning interest in animal-microbiota interactions, but dissecting the specific impacts of microbes on their hosts is experimentally challenging. Gnotobiology, the study of biological systems in which all members are known, enables precise experimental analysis of the necessity and sufficiency of microbes in animal biology by deriving animals germ-free (GF) and inoculating them with defined microbial lineages. Mammalian host models have long dominated gnotobiology, but we have recently adapted gnotobiotic approaches to the zebrafish (Danio rerio), an important aquatic model. Zebrafish offer several experimental attributes that enable rapid, large-scale gnotobiotic experimentation with high replication rates and exquisite optical resolution. Here we describe detailed protocols for three procedures that form the foundation of zebrafish gnotobiology: derivation of GF embryos, microbial association of GF animals, and long-term, GF husbandry. Our aim is to provide sufficient guidance in zebrafish gnotobiotic methodology to expand and enrich this exciting field of research.