RESUMO
Understanding and predicting whether new drug candidates will be safe in the clinic is a critical hurdle in pharmaceutical development, that relies in part on absorption, distribution, metabolism, excretion and toxicology studies in vivo. Zebrafish is a relatively new model system for drug metabolism and toxicity studies, offering whole organism screening coupled with small size and potential for high-throughput screening. Through toxicity and absorption analyses of a number of drugs, we find that zebrafish is generally predictive of drug toxicity, although assay outcomes are influenced by drug lipophilicity which alters drug uptake. In addition, liver microsome assays reveal specific differences in metabolism of compounds between human and zebrafish livers, likely resulting from the divergence of the cytochrome P450 superfamily between species. To reflect human metabolism more accurately, we generated a transgenic "humanized" zebrafish line that expresses the major human phase I detoxifying enzyme, CYP3A4, in the liver. Here, we show that this humanized line shows an elevated metabolism of CYP3A4-specific substrates compared to wild-type zebrafish. The generation of this first described humanized zebrafish liver suggests such approaches can enhance the accuracy of the zebrafish model for toxicity prediction.
Assuntos
Citocromo P-450 CYP3A/genética , Fígado/efeitos dos fármacos , Farmacocinética , Testes de Toxicidade/métodos , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Cromatografia Líquida de Alta Pressão , Citocromo P-450 CYP3A/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Inativação Metabólica , Fígado/metabolismo , Espectrometria de Massas , Preparações Farmacêuticas/química , SolubilidadeRESUMO
Developmental patterning and growth of the vertebrate digestive and respiratory tracts requires interactions between the epithelial endoderm and adjacent mesoderm. The esophagus is a specialized structure that connects the digestive and respiratory systems and its normal development is critical for both. Shh signaling from the epithelium regulates related aspects of mammalian and zebrafish digestive organ development and has a prominent effect on esophageal morphogenesis. The mechanisms underlying esophageal malformations, however, are poorly understood. Here, we show that zebrafish Ihha signaling from the epithelium acting in parallel, but independently of Shh, controls epithelial and mesenchymal cell proliferation and differentiation of smooth muscles and neurons in the gut and swimbladder. In zebrafish ihha mutants, the esophageal and swimbladder epithelium is dysmorphic, and expression of fgf10 in adjacent mesenchymal cells is affected. Analysis of the development of the esophagus and swimbladder in fgf10 mutant daedalus (dae) and compound dae/ihha mutants shows that the Ihha-Fgf10 regulatory interaction is realized through a signaling feedback loop between the Ihha-expressing epithelium and Fgf10-expressing mesenchyme. Disruption of this loop further affects the esophageal and swimbladder epithelium in ihha mutants, and Ihha acts in parallel to but independently of Shha in this process. These findings contribute to the understanding of epithelial-mesenchymal interactions and highlight an interaction between Hh and Fgf signaling pathways during esophagus and swimbladder development.
Assuntos
Fator 10 de Crescimento de Fibroblastos/metabolismo , Proteínas Hedgehog/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Sacos Aéreos/embriologia , Sacos Aéreos/metabolismo , Animais , Animais Geneticamente Modificados , Proliferação de Células , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Epitélio/embriologia , Epitélio/metabolismo , Esôfago/embriologia , Esôfago/metabolismo , Feminino , Fator 10 de Crescimento de Fibroblastos/genética , Trato Gastrointestinal/citologia , Trato Gastrointestinal/embriologia , Trato Gastrointestinal/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas Hedgehog/genética , Hibridização In Situ , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mesoderma/embriologia , Mesoderma/metabolismo , Microscopia Confocal , Mutação , Receptores Patched , Ligação Proteica , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Transdução de Sinais/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
Zebrafish are increasingly used to study neurodegenerative conditions such as Parkinson's disease (PD). In rodents, the influence of the genetic background on important experimental parameters in PD research such as susceptibility to toxin exposure or motor behavior is well established. In contrast, little is known about the impact of the genetic background in commonly used zebrafish wild-type strains on these important experimental parameters. We determined the effect of the genetic background in five commonly used zebrafish wild-type strains on crucial, PD-related aspects, in particular the number of ascending dopaminergic neurons, their susceptibility to PD-related neurotoxins, and the expression levels of five genes involved in oxidative stress defense, protein degradation, cell death, and apoptosis. We also investigated whether the susceptibility to morpholino-mediated knockdown of the PD gene DJ-1 may have a varying effect on neuronal cell loss depending on the genetic background. Finally, we determined the influence of the genetic background on spontaneous motor behavior. There was remarkably little variation between the different wild-type strains for most parameters investigated. However, the susceptibility to the neurotoxin 1-methyl-4-phenylpyridinium differed between the five investigated strains and so did their spontaneous motor behavior.
Assuntos
Doença de Parkinson/genética , Peixe-Zebra/genética , Animais , Contagem de Células , Modelos Animais de Doenças , Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Morfolinos/genética , Atividade Motora/efeitos dos fármacos , Atividade Motora/genética , Proteínas do Tecido Nervoso/genética , Neurotoxinas/farmacologia , Doença de Parkinson/metabolismo , Proteínas de Peixe-Zebra/genéticaRESUMO
Using forward genetics, we have identified the genes mutated in two classes of zebrafish fin mutants. The mutants of the first class are characterized by defects in embryonic fin morphogenesis, which are due to mutations in a Laminin subunit or an Integrin alpha receptor, respectively. The mutants of the second class display characteristic blistering underneath the basement membrane of the fin epidermis. Three of them are due to mutations in zebrafish orthologues of FRAS1, FREM1, or FREM2, large basement membrane protein encoding genes that are mutated in mouse bleb mutants and in human patients suffering from Fraser Syndrome, a rare congenital condition characterized by syndactyly and cryptophthalmos. Fin blistering in a fourth group of zebrafish mutants is caused by mutations in Hemicentin1 (Hmcn1), another large extracellular matrix protein the function of which in vertebrates was hitherto unknown. Our mutant and dose-dependent interaction data suggest a potential involvement of Hmcn1 in Fraser complex-dependent basement membrane anchorage. Furthermore, we present biochemical and genetic data suggesting a role for the proprotein convertase FurinA in zebrafish fin development and cell surface shedding of Fras1 and Frem2, thereby allowing proper localization of the proteins within the basement membrane of forming fins. Finally, we identify the extracellular matrix protein Fibrillin2 as an indispensable interaction partner of Hmcn1. Thus we have defined a series of zebrafish mutants modelling Fraser Syndrome and have identified several implicated novel genes that might help to further elucidate the mechanisms of basement membrane anchorage and of the disease's aetiology. In addition, the novel genes might prove helpful to unravel the molecular nature of thus far unresolved cases of the human disease.
Assuntos
Embrião não Mamífero/metabolismo , Proteínas da Matriz Extracelular/genética , Síndrome de Frasier/genética , Furina/genética , Mutação , Pró-Proteína Convertases/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/embriologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas da Matriz Extracelular/metabolismo , Furina/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Dados de Sequência Molecular , Pró-Proteína Convertases/metabolismo , Proteínas de Peixe-Zebra/metabolismoRESUMO
Currently, only symptomatic therapy is available for Parkinson's disease. The zebrafish is a vertebrate animal model ideally suited for high throughput compound screening to identify disease-modifying compounds for Parkinson's disease. We have developed a zebrafish model for Parkin deficiency, the most commonly mutated gene in early onset Parkinson's disease. The zebrafish Parkin protein is 62% identical to its human counterpart with 78% identity in functionally relevant regions. The parkin gene is expressed throughout zebrafish development and ubiquitously in adult zebrafish tissue. Abrogation of Parkin activity leads to a significant decrease in the number of ascending dopaminergic neurons in the posterior tuberculum (homologous to the substantia nigra in humans), an effect enhanced by exposure to MPP+. Both light microscopic analysis and staining with the pan-neuronal marker HuC confirmed that this loss of dopaminergic neurons is not due to general impairment of brain development. Neither serotonergic nor motor neurons were affected, further emphasizing that the effect of parkin knockdown appears to be specific for dopaminergic neurons. Notably, parkin knockdown zebrafish embryos also develop specific reduction in the activity of the mitochondrial respiratory chain complex I, making this the first vertebrate model to share both important pathogenic mechanisms (i.e. complex I deficiency) and the pathological hallmark (i.e. dopaminergic cell loss) with human parkin-mutant patients. The zebrafish model is thus ideally suited for future drug screens and other studies investigating the functional mechanisms underlying neuronal cell death in early onset Parkinson's Disease. Additional electron microscopy studies revealed electron dense material in the t-tubules within the muscle tissue of parkin knockdown zebrafish. T-tubules are rich in L-type calcium channels, therefore our work might also provide a tentative link between genetically determined early onset Parkinson's disease and recent studies attributing an important role to these L-type calcium channels in late onset sporadic Parkinson's disease.
Assuntos
Modelos Animais de Doenças , Complexo I de Transporte de Elétrons/deficiência , Doença de Parkinson/metabolismo , Ubiquitina-Proteína Ligases/deficiência , Animais , Sequência Conservada , Dopamina/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Desenvolvimento Embrionário/fisiologia , Éxons/genética , Técnicas de Silenciamento de Genes , Microscopia Eletrônica , Mitocôndrias Musculares/ultraestrutura , Doenças Mitocondriais/genética , Neurônios/patologia , Oligonucleotídeos Antissenso , Doença de Parkinson/genética , Doença de Parkinson/patologia , Sítios de Splice de RNA/genética , Substância Negra/patologia , Natação , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Peixe-ZebraRESUMO
The zebrafish, long recognized as a model organism for the analysis of basic developmental processes, is now also emerging as an alternative animal model for human diseases. This review will first provide an overview of the particular characteristics of zebrafish in general and their dopaminergic nervous system in particular. We will then summarize all work undertaken so far to establish zebrafish as a new animal model for movement disorders and will finally emphasize its particular strength - amenability to high throughput in vivo drug screening.
Assuntos
Antiparkinsonianos/farmacologia , Modelos Animais de Doenças , Doença de Huntington/genética , Transtornos dos Movimentos/genética , Doença de Parkinson/genética , Peixe-Zebra/genética , Animais , Avaliação Pré-Clínica de Medicamentos , Predisposição Genética para Doença/genética , Doença de Huntington/tratamento farmacológico , Doença de Huntington/fisiopatologia , Transtornos dos Movimentos/tratamento farmacológico , Transtornos dos Movimentos/fisiopatologia , Mutação/genética , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/fisiopatologia , Peixe-Zebra/metabolismoRESUMO
Mutations in DJ-1 lead to early onset Parkinson's disease (PD). The aim of this study was to elucidate further the underlying mechanisms leading to neuronal cell death in DJ-1 deficiency in vivo and determine whether the observed cell loss could be prevented pharmacologically. Inactivation of DJ-1 in zebrafish, Danio rerio, resulted in loss of dopaminergic neurons after exposure to hydrogen peroxide and the proteasome inhibitor MG132. DJ-1 knockdown by itself already resulted in increased p53 and Bax expression levels prior to toxin exposure without marked neuronal cell death, suggesting subthreshold activation of cell death pathways in DJ-1 deficiency. Proteasome inhibition led to a further increase of p53 and Bax expression with widespread neuronal cell death. Pharmacological p53 inhibition either before or during MG132 exposure in vivo prevented dopaminergic neuronal cell death in both cases. Simultaneous knockdown of DJ-1 and the negative p53 regulator mdm2 led to dopaminergic neuronal cell death even without toxin exposure, further implicating involvement of p53 in DJ-1 deficiency-mediated neuronal cell loss. Our study demonstrates the utility of zebrafish as a new animal model to study PD gene defects and suggests that modulation of downstream mechanisms, such as p53 inhibition, may be of therapeutic benefit.
Assuntos
Proteínas do Tecido Nervoso/deficiência , Neurônios/fisiologia , Doença de Parkinson/patologia , Proteína Supressora de Tumor p53/fisiologia , Proteínas de Peixe-Zebra/deficiência , Animais , Animais Geneticamente Modificados , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Modelos Animais de Doenças , Embrião de Mamíferos , Embrião não Mamífero , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Peróxido de Hidrogênio/farmacologia , Hibridização In Situ/métodos , Marcação In Situ das Extremidades Cortadas , Leupeptinas/farmacologia , Neurônios/efeitos dos fármacos , Neurotoxinas/farmacologia , Doença de Parkinson/genética , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Tirosina 3-Mono-Oxigenase/metabolismo , Peixe-Zebra , Proteína X Associada a bcl-2/metabolismoRESUMO
Signalling by members of the Hedgehog family of secreted proteins plays a central role in the development of many animal species. In the zebrafish embryo, the specification of myoblast fates is controlled by Hedgehog signals emanating from axial midline structures. Distinct muscle cell identities are induced by varying levels of signalling activity. The SET domain transcription factor, Blimp1, is a key target of Hedgehog signalling in this process.
Assuntos
Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Músculo Esquelético/embriologia , Transdução de Sinais/fisiologia , Transativadores/fisiologia , Peixe-Zebra/embriologia , Animais , Diferenciação Celular , Proteínas de Ligação a DNA , Indução Embrionária , Proteínas Hedgehog , Proteínas Nucleares , Fator 1 de Ligação ao Domínio I Regulador Positivo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Peixe-ZebraRESUMO
Members of the T-box (tbx) gene family encode developmentally regulated transcription factors, several of which are implicated in human hereditary diseases. We have cloned the paralogous genes tbx15 and tbx18 in zebrafish and have characterised their expression in detail. tbx15 is expressed in paraxial head mesenchyme and its derivatives, the extraocular and jaw musculature and the posterior neurocranium. Further areas of tbx15 expression are in the anterior somitic mesoderm, in periocular mesenchyme and in the pectoral fin mesenchyme throughout larval development. Areas of strong tbx18 expression are found in the developing somitic and presomitic mesoderm, in the heart and in pectoral fin mesenchyme, as well as the ventral neuroectoderm and the developing palate. Both genes exhibit particular differences in expression compared to their murine orthologs.