RESUMO
Parkinson's disease (PD) is a neurodegenerative disorder that is clinically assessed by motor symptoms associated with the loss of midbrain dopaminergic neurons affecting the quality of life for over 8.5 million people worldwide. The neurotoxin 6-hydroxydopamine (6-OHDA) has been used to chemically induce a PD-like state in zebrafish larvae by several laboratories; however, highly variable concentration, methodology, and reagents have resulted in conflicting results suggesting a need to investigate these issues of reproducibility. We propose a protocol that addresses the differences in methodology and induces changes in 6 days postfertilization (dpf) larvae utilizing a 24-h exposure at 3 dpf with 30 µM 6-OHDA. Despite â¼50% lethality, no morphological or development differences in surviving fish are observed. Definition of our model is defined by downregulation of the expression of th1 by reverse transcriptase-quantitative polymerase chain reaction, a marker for dopaminergic neurons and a reduction in movement. Additionally, we observed a downregulation of pink1 and an upregulation of sod1 and sod2, indicators of mitochondrial dysfunction and response to reactive oxygen species, respectively.
Assuntos
Neurônios Dopaminérgicos , Larva , Oxidopamina , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Peixe-Zebra/genética , Oxidopamina/farmacologia , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Larva/crescimento & desenvolvimento , Larva/efeitos dos fármacos , Larva/genética , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Neurotoxinas/toxicidade , Modelos Animais de Doenças , Superóxido Dismutase/metabolismo , Superóxido Dismutase/genética , Proteínas Serina-Treonina QuinasesRESUMO
Zebrafish larvae have several biological features that make them useful for cellular investigations of the mechanisms underlying learning and memory. Of particular interest in this regard is a rapid escape, or startle, reflex possessed by zebrafish larvae; this reflex, the C-start, is mediated by a relatively simple neuronal circuit and exhibits habituation, a non-associative form of learning. Here we demonstrate a rapid form of habituation of the C-start to touch that resembles the previously reported rapid habituation induced by auditory or vibrational stimuli. We also show that touch-induced habituation exhibits input specificity. This work sets the stage for in vivo optical investigations of the cellular sites of plasticity that mediate habituation of the C-start in the larval zebrafish.
Assuntos
Reação de Fuga/fisiologia , Habituação Psicofisiológica , Tato/fisiologia , Peixe-Zebra/fisiologia , Animais , Eletrochoque , Reação de Fuga/efeitos dos fármacos , Glicina/farmacologia , Habituação Psicofisiológica/efeitos dos fármacos , Cabeça , Larva/efeitos dos fármacos , Larva/fisiologia , Reflexo de Sobressalto/efeitos dos fármacos , Reflexo de Sobressalto/fisiologia , Estricnina/farmacologiaRESUMO
The cellular and molecular basis of long-term memory in vertebrates remains poorly understood. Knowledge regarding long-term memory has been impeded by the enormous complexity of the vertebrate brain, particularly the mammalian brain, as well as by the relative complexity of the behavioral alterations examined in most studies of long-term memory in vertebrates. Here, we demonstrate a long-term form of nonassociative learning-specifically, long-term habituation (LTH)-of a simple reflexive escape response, the C-start, in zebrafish larvae. The C-start is triggered by the activation of one of a pair of giant neurons in the zebrafish's hindbrain, the Mauthner cells. We show that LTH of the C-start requires the activity of NMDA receptors and involves macromolecular synthesis. We further show that the long-term habituated reflex can by rapidly dishabituated by a brief tactile stimulus. Our results set the stage for rigorous, mechanistic investigations of the long-term memory for habituation of a reflexive behavioral response, one that is mediated by a relatively simple, neurobiologically tractable, neural circuit. Moreover, the demonstration of NMDAR and transcriptionally dependent LTH in a translucent vertebrate organism should facilitate the use of optical recording, and optogenetic manipulation, of neuronal activity to elucidate the cellular basis of a long-term vertebrate memory.
Assuntos
Habituação Psicofisiológica/fisiologia , Receptores de N-Metil-D-Aspartato/fisiologia , Reflexo/fisiologia , Peixe-Zebra/fisiologia , Animais , Larva , Proteínas de Peixe-ZebraRESUMO
Autism spectrum disorders (ASDs) are a group of devastating neurodevelopmental syndromes that affect up to 1 in 68 children. Despite advances in the identification of ASD risk genes, the mechanisms underlying ASDs remain unknown. Homozygous loss-of-function mutations in Contactin Associated Protein-like 2 (CNTNAP2) are strongly linked to ASDs. Here we investigate the function of Cntnap2 and undertake pharmacological screens to identify phenotypic suppressors. We find that zebrafish cntnap2 mutants display GABAergic deficits, particularly in the forebrain, and sensitivity to drug-induced seizures. High-throughput behavioral profiling identifies nighttime hyperactivity in cntnap2 mutants, while pharmacological testing reveals dysregulation of GABAergic and glutamatergic systems. Finally, we find that estrogen receptor agonists elicit a behavioral fingerprint anti-correlative to that of cntnap2 mutants and show that the phytoestrogen biochanin A specifically reverses the mutant behavioral phenotype. These results identify estrogenic compounds as phenotypic suppressors and illuminate novel pharmacological pathways with relevance to autism.
Assuntos
Transtorno Autístico/tratamento farmacológico , Estrogênios/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Proteínas de Membrana/genética , Mutação/genética , Proteínas do Tecido Nervoso/genética , Animais , Animais Geneticamente Modificados , Transtorno Autístico/genética , Modelos Animais de Doenças , Estrogênios/uso terapêutico , Genisteína/farmacologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Larva , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Atividade Motora/efeitos dos fármacos , Atividade Motora/genética , Fenótipo , Fitoestrógenos/farmacologia , Psicotrópicos/farmacologia , Psicotrópicos/uso terapêutico , Convulsões/tratamento farmacológico , Convulsões/genética , Transtornos da Transição Sono-Vigília/tratamento farmacológico , Transtornos da Transição Sono-Vigília/genética , Proteína Vesicular 2 de Transporte de Glutamato/genética , Proteína Vesicular 2 de Transporte de Glutamato/metabolismo , Peixe-ZebraRESUMO
Neurodevelopmental and neuropsychiatric disorders result from complex interactions between critical genetic factors and as-yet-unknown environmental components. To gain clinical insight, it is critical to develop a comprehensive understanding of these genetic components. RBFOX1, an RNA splicing factor, regulates expression of large genetic networks during early neuronal development, and haploinsufficiency causes severe neurodevelopmental phenotypes including autism spectrum disorder (ASD), intellectual disability, and epilepsy. Genomic testing in individuals and large patient cohorts has identified phenotypically similar cases possessing copy number variations in RBFOX1, implicating the gene as an important cause of neurodevelopmental disease. However, a significant proportion of the observed structural variation is inherited from phenotypically normal individuals, raising questions regarding overall pathogenicity of variation at the RBFOX1 locus. In this chapter, we discuss the molecular, cellular, and clinical evidence supporting the role of RBFOX1 in neurodevelopment and present a comprehensive model for the contribution of structural variation in RBFOX1 to ASD.
Assuntos
Transtornos Globais do Desenvolvimento Infantil/genética , Variações do Número de Cópias de DNA/genética , Deficiências do Desenvolvimento/patologia , Proteínas de Ligação a RNA/genética , Animais , Deficiências do Desenvolvimento/metabolismo , Epilepsia/genética , Humanos , Deficiência Intelectual/genética , Fatores de Processamento de RNA , Proteínas de Ligação a RNA/metabolismoRESUMO
Larval zebrafish possess several experimental advantages for investigating the molecular and neural bases of learning and memory. Despite this, neuroscientists have only recently begun to use these animals to study memory. However, in a relatively short period of time a number of forms of learning have been described in zebrafish larvae, and significant progress has been made toward their understanding. Here we provide a comprehensive review of this progress; we also describe several promising new experimental technologies currently being used in larval zebrafish that are likely to contribute major insights into the processes that underlie learning and memory.
Assuntos
Habituação Psicofisiológica/fisiologia , Memória/fisiologia , Animais , Humanos , Larva , Aprendizagem/fisiologia , Peixe-ZebraRESUMO
The Homeobox (Hox) and Paired box (Pax) gene families are key determinants of animal body plans and organ structure. In particular, they function within regulatory networks that control organogenesis. How these conserved genes elicit differences in organ form and function in response to evolutionary pressures is incompletely understood. We molecularly and functionally characterized one member of an evolutionarily dynamic gene family, plac8 onzin related protein 1 (ponzr1), in the zebrafish. ponzr1 mRNA is expressed early in the developing kidney and pharyngeal arches. Using ponzr1-targeting morpholinos, we show that ponzr1 is required for formation of the glomerulus. Loss of ponzr1 results in a nonfunctional glomerulus but retention of a functional pronephros, an arrangement similar to the aglomerular kidneys found in a subset of marine fish. ponzr1 is integrated into the pax2a pathway, with ponzr1 expression requiring pax2a gene function, and proper pax2a expression requiring normal ponzr1 expression. In addition to pronephric function, ponzr1 is required for pharyngeal arch formation. We functionally demonstrate that ponzr1 can act as a transcription factor or co-factor, providing the first molecular mode of action for this newly described gene family. Together, this work provides experimental evidence of an additional mechanism that incorporates evolutionarily dynamic, lineage-specific gene families into conserved regulatory gene networks to create functional organ diversity.
Assuntos
Região Branquial/embriologia , Pronefro/embriologia , Fatores de Transcrição/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/anatomia & histologia , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Evolução Biológica , Biomarcadores/metabolismo , Região Branquial/metabolismo , Embrião não Mamífero/anatomia & histologia , Embrião não Mamífero/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Rim/anatomia & histologia , Morfogênese/fisiologia , Fator de Transcrição PAX2/genética , Fator de Transcrição PAX2/metabolismo , Fenótipo , Pronefro/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Peixe-Zebra/metabolismoRESUMO
This study describes the expression of the pituitary adenylate cyclase-activating polypeptide (PACAP1 and PACAP2) and PAC1 receptor genes (PAC1a-R and PAC1b-R) in the brain of zebrafish (Danio rerio) during development. In situ hybridization of the 24- and 48-hpf embryos revealed that PACAP genes were expressed in the telencephalon, the diencephalon, the rhombencephalon, and the neurons in the dorsal part of the spinal cord. PACAP2 mRNA appears to be the most abundant form during brain development. The two PAC1-R subtypes showed a similar expression pattern: mRNAs were detected in the forebrain, the thalamus, and the rhombencephalon. However, in the tectum, only PAC1b-R gene was detected. These results suggest that, in fish, PACAP may play a role in brain development.
Assuntos
Embrião não Mamífero/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Animais , Encéfalo/embriologia , Encéfalo/metabolismo , Embrião não Mamífero/anatomia & histologia , Expressão Gênica , Hibridização In Situ , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Peixe-Zebra/metabolismoRESUMO
The autism spectrum disorders (ASD) are a heterogeneous set of developmental disorders characterized at their core by deficits in social interaction and communication. Current psychiatric nosology groups this broad set of disorders with strong genetic liability and multiple etiologies into the same diagnostic category. This heterogeneity has challenged genetic analyses. But shared patient resources, genomic technologies, more refined phenotypes, and novel computational approaches have begun to yield dividends in defining the genetic mechanisms at work. Over the last five years, a large number of autism susceptibility loci have emerged, redefining our notion of autism's etiologies, and reframing how we think about ASD.
Assuntos
Transtorno Autístico/genética , Heterogeneidade Genética , Predisposição Genética para Doença/genética , Transdução de Sinais/genética , Transtorno Autístico/patologia , Epigênese Genética , Redes Reguladoras de Genes , Humanos , Modelos GenéticosRESUMO
Morpholino oligonucleotides are the most common anti-sense "knockdown" technique used in zebrafish (Danio rerio). This review discusses common practices for the design, preparation, and deployment of morpholinos in this vertebrate model system. Off-targeting effects of morpholinos are discussed as well as method to minimize this potentially confounding variable via co-injection of a tP53-targeting morpholino. Finally, new uses of morpholinos are summarized and contextualized with respect to the complementary, DNA-based knockout technologies recently developed for zebrafish.
Assuntos
Técnicas de Silenciamento de Genes , Oligonucleotídeos/genética , Peixe-Zebra/genética , AnimaisRESUMO
BACKGROUND: The choroid plexus (CP) is an epithelial and vascular structure in the ventricular system of the brain that is a critical part of the blood-brain barrier. The CP has two primary functions, 1) to produce and regulate components of the cerebral spinal fluid, and 2) to inhibit entry into the brain of exogenous substances. Despite its importance in neurobiology, little is known about how this structure forms. METHODOLOGY AND PRINCIPAL FINDINGS: Here we show that the transposon-mediated enhancer trap zebrafish line Et(Mn16) expresses green fluorescent protein within a population of cells that migrate toward the midline and coalesce to form the definitive CP. We further demonstrate the development of the integral vascular network of the definitive CP. Utilizing pharmacologic pan-notch inhibition and specific morpholino-mediated knockdown, we demonstrate a requirement for Notch signaling in choroid plexus development. We identify three Notch signaling pathway members as mediating this effect, notch1b, deltaA, and deltaD. CONCLUSIONS AND SIGNIFICANCE: This work is the first to identify the zebrafish choroid plexus and to characterize its epithelial and vasculature integration. This study, in the context of other comparative anatomical studies, strongly indicates a conserved mechanism for development of the CP. Finally, we characterize a requirement for Notch signaling in the developing CP. This establishes the zebrafish CP as an important new system for the determination of key signaling pathways in the formation of this essential component of the vertebrate brain.
Assuntos
Plexo Corióideo/embriologia , Plexo Corióideo/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Receptores Notch/metabolismo , Animais , Transporte Biológico , Barreira Hematoencefálica , Encéfalo/metabolismo , Epitélio/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Microscopia de Fluorescência , Modelos Biológicos , Transdução de Sinais , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismoRESUMO
BACKGROUND: The zebrafish is a powerful model vertebrate amenable to high throughput in vivo genetic analyses. Examples include reverse genetic screens using morpholino knockdown, expression-based screening using enhancer trapping and forward genetic screening using transposon insertional mutagenesis. We have created a database to facilitate web-based distribution of data from such genetic studies. DESCRIPTION: The MOrpholino DataBase is a MySQL relational database with an online, PHP interface. Multiple quality control levels allow differential access to data in raw and finished formats. MODBv1 includes sequence information relating to almost 800 morpholinos and their targets and phenotypic data regarding the dose effect of each morpholino (mortality, toxicity and defects). To improve the searchability of this database, we have incorporated a fixed-vocabulary defect ontology that allows for the organization of morpholino affects based on anatomical structure affected and defect produced. This also allows comparison between species utilizing Phenotypic Attribute Trait Ontology (PATO) designated terminology. MODB is also cross-linked with ZFIN, allowing full searches between the two databases. MODB offers users the ability to retrieve morpholino data by sequence of morpholino or target, name of target, anatomical structure affected and defect produced. CONCLUSION: MODB data can be used for functional genomic analysis of morpholino design to maximize efficacy and minimize toxicity. MODB also serves as a template for future sequence-based functional genetic screen databases, and it is currently being used as a model for the creation of a mutagenic insertional transposon database.
Assuntos
Elementos Antissenso (Genética) , Sistemas de Gerenciamento de Base de Dados , Bases de Dados Genéticas , Interface Usuário-Computador , Peixe-Zebra/genética , Animais , Mapeamento Cromossômico/métodos , Biologia Computacional , Embrião não Mamífero , Expressão Gênica , Perfilação da Expressão Gênica , Genômica , Internet , Oligonucleotídeos , Fenótipo , Especificidade da Espécie , Vocabulário Controlado , Peixe-Zebra/embriologiaRESUMO
BACKGROUND: The retinal vasculature is a capillary network of blood vessels that nourishes the inner retina of most mammals. Developmental abnormalities or microvascular complications in the retinal vasculature result in severe human eye diseases that lead to blindness. To exploit the advantages of zebrafish for genetic, developmental and pharmacological studies of retinal vasculature, we characterised the intraocular vasculature in zebrafish. RESULTS: We show a detailed morphological and developmental analysis of the retinal blood supply in zebrafish. Similar to the transient hyaloid vasculature in mammalian embryos, vessels are first found attached to the zebrafish lens at 2.5 days post fertilisation. These vessels progressively lose contact with the lens and by 30 days post fertilisation adhere to the inner limiting membrane of the juvenile retina. Ultrastructure analysis shows these vessels to exhibit distinctive hallmarks of mammalian retinal vasculature. For example, smooth muscle actin-expressing pericytes are ensheathed by the basal lamina of the blood vessel, and vesicle vacuolar organelles (VVO), subcellular mediators of vessel-retinal nourishment, are present. Finally, we identify 9 genes with cell membrane, extracellular matrix and unknown identity that are necessary for zebrafish hyaloid and retinal vasculature development. CONCLUSION: Zebrafish have a retinal blood supply with a characteristic developmental and adult morphology. Abnormalities of these intraocular vessels are easily observed, enabling application of genetic and chemical approaches in zebrafish to identify molecular regulators of hyaloid and retinal vasculature in development and disease.
Assuntos
Corioide/irrigação sanguínea , Regulação da Expressão Gênica no Desenvolvimento , Vasos Retinianos/crescimento & desenvolvimento , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/genética , Animais , Corioide/crescimento & desenvolvimento , Proteínas Contráteis/genética , Embrião não Mamífero , Proteínas da Matriz Extracelular/genética , Humanos , Imuno-Histoquímica , Neovascularização Fisiológica/genética , Disco Óptico/irrigação sanguínea , Fatores de Processamento de RNA , Receptores de Superfície Celular/genética , Neovascularização Retiniana/genética , Especificidade da Espécie , Proteínas de Peixe-Zebra/genéticaRESUMO
Low-copy repeats (LCRs) are genomic features that affect chromosome stability and can produce disease-associated rearrangements. We describe members of three families with deletions in 10q22.3-q23.31, a region harboring a complex set of LCRs, and demonstrate that rearrangements in this region are associated with behavioral and neurodevelopmental abnormalities, including cognitive impairment, autism, hyperactivity, and possibly psychiatric disease. Fine mapping of the deletions in members of all three families by use of a custom 10q oligonucleotide array-based comparative genomic hybridization (NimbleGen) and polymerase chain reaction-based methods demonstrated a different deletion in each family. In one proband, the deletion breakpoints are associated with DNA fragments containing noncontiguous sequences of chromosome 10, whereas, in the other two families, the breakpoints are within paralogous LCRs, removing approximately 7.2 Mb and 32 genes. Our data provide evidence that the 10q22-q23 genomic region harbors one or more genes important for cognitive and behavioral development and that recurrent deletions affecting this interval define a novel genomic disorder.
Assuntos
Transtornos do Comportamento Infantil/genética , Deleção Cromossômica , Cromossomos Humanos Par 10/genética , Transtornos Cognitivos/genética , Transtorno Autístico/genética , Criança , Pré-Escolar , Bandeamento Cromossômico , Quebra Cromossômica , Mapeamento Cromossômico , Cromossomos Artificiais Bacterianos/genética , Feminino , Rearranjo Gênico , Humanos , Hibridização in Situ Fluorescente , Lactente , Masculino , Modelos Genéticos , Análise de Sequência com Séries de Oligonucleotídeos , Linhagem , Sequências Repetitivas de Ácido NucleicoRESUMO
BACKGROUND: Understanding the functional role(s) of the more than 20,000 proteins of the vertebrate genome is a major next step in the post-genome era. The approximately 4,000 co-translationally translocated (CTT) proteins - representing the vertebrate secretome - are important for such vertebrate-critical processes as organogenesis. However, the role(s) for most of these genes is currently unknown. RESULTS: We identified 585 putative full-length zebrafish CTT proteins using cross-species genomic and EST-based comparative sequence analyses. We further investigated 150 of these genes (Figure 1) for unique function using morpholino-based analysis in zebrafish embryos. 12% of the CTT protein-deficient embryos resulted in specific developmental defects, a notably higher rate of gene function annotation than the 2%-3% estimate from random gene mutagenesis studies. CONCLUSION: This initial collection includes novel genes required for the development of vascular, hematopoietic, pigmentation, and craniofacial tissues, as well as lipid metabolism, and organogenesis. This study provides a framework utilizing zebrafish for the systematic assignment of biological function in a vertebrate genome.