Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 5.805
Filtrar
1.
Zhong Nan Da Xue Xue Bao Yi Xue Ban ; 44(9): 968-975, 2019 Sep 28.
Artigo em Chinês | MEDLINE | ID: mdl-31645484

RESUMO

OBJECTIVE: To explore the effects of KIAA0196 gene on cardiac development and the establishment of zebrafish strain.
 Methods: Peripheral blood and gDNA from patients were extracted. Copy number variation analysis and target sequencing were conducted to screen candidate genes. The KIAA0196 knockout zebrafish was generated by CRISPR/Cas9 to detect whether KIAA0196 deficiency could affect cardiac development. Finally, the wild-type and mutant zebrafish were anatomized and histologically stained to observe the phenotype of heart defects.
 Results: The KIAA0196 knockout zebrafish strain was successfully constructed using CRISPR/Cas9 technology. After 60 hours fertilization, microscopic examination of KIAA0196 knockout zebrafish (heterozygote + homozygote) showed pericardial effusion, cardiac compression and severely curly tail. Compared with wild-type zebrafish, the hearts of mutant KIAA0196 zebrafish had cardiac defects including smaller atrium and larger ventricle, and the myocardial cells were looser.
 Conclusion: KIAA0196 gene plays an important regulatory role in the development of heart. It might be a candidate gene for congenital heart disease.


Assuntos
Cardiopatias Congênitas/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Variações do Número de Cópias de DNA , Coração , Humanos , Miócitos Cardíacos , Fenótipo , Proteínas
2.
Toxicol Lett ; 314: 43-52, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31310794

RESUMO

Thioredoxin is an evolutionarily conserved antioxidant protein that plays a crucial role for fundamental cellular processes and embryonic development. Growing evidence support that Thioredoxin influences cellular response to chemicals insults, particularly those accompanying oxidative stress. The mechanisms underlying the functions of Thioredoxin1 in the embryonic development under the environmental toxicant exposure remain, however, largely unexplored. We report here that thioredoxin1 becomes differentially expressed in zebrafish embryos after exposure to 9 out of 11 environmental chemicals. In situ gene expression analysis show that thioredoxin1 is expressed in neurons, olfactory epithelia, liver and swim bladder under normal conditions. After MeHg exposure, however, thioredoxin1 is ectopically induced in the hair cells of the lateral line and in epithelia cells of the pharynx. Knockdown of Thioredoxin1 induces hydrocephalus and increases cell apoptosis in the brain ventricular epithelia cells. In comparison with 5% malformation in embryos injected with control morpholino, MeHg induces more than 77% defects in Thioredoxin1 knockdown embryos. Our data suggest that there is an association between hydrocephalus and Thioredoxin1 malfunction in embryonic development, and provide valuable information to elucidate the protective role of Thioredoxin1 against chemicals disruption.


Assuntos
Encéfalo/efeitos dos fármacos , Poluentes Ambientais/toxicidade , Hidrocefalia/induzido quimicamente , Tiorredoxinas/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Apoptose/efeitos dos fármacos , Encéfalo/embriologia , Encéfalo/metabolismo , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Embrião não Mamífero/patologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Regulação da Expressão Gênica no Desenvolvimento , Hidrocefalia/embriologia , Hidrocefalia/genética , Hidrocefalia/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Estresse Oxidativo/efeitos dos fármacos , Tiorredoxinas/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
3.
Nat Commun ; 10(1): 3049, 2019 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-31296872

RESUMO

The transcription factor p63 is a master regulator of ectoderm development. Although previous studies show that p63 triggers epidermal differentiation in vitro, the roles of p63 in developing embryos remain poorly understood. Here, we use zebrafish embryos to analyze in vivo how p63 regulates gene expression during development. We generate tp63-knock-out mutants that recapitulate human phenotypes and show down-regulated epidermal gene expression. Following p63-binding dynamics, we find two distinct functions clearly separated in space and time. During early development, p63 binds enhancers associated to neural genes, limiting Sox3 binding and reducing neural gene expression. Indeed, we show that p63 and Sox3 are co-expressed in the neural plate border. On the other hand, p63 acts as a pioneer factor by binding non-accessible chromatin at epidermal enhancers, promoting their opening and epidermal gene expression in later developmental stages. Therefore, our results suggest that p63 regulates cell fate decisions during vertebrate ectoderm specification.


Assuntos
Ectoderma/embriologia , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Placa Neural/embriologia , Fosfoproteínas/metabolismo , Transativadores/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Sistemas CRISPR-Cas/genética , Diferenciação Celular/genética , Cromatina/metabolismo , Regulação para Baixo , Ectoderma/metabolismo , Embrião não Mamífero , Elementos Facilitadores Genéticos/genética , Epiderme/embriologia , Epiderme/metabolismo , Técnicas de Inativação de Genes , Modelos Animais , Placa Neural/metabolismo , Fosfoproteínas/genética , Ligação Proteica/genética , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Transativadores/genética , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/genética
4.
Aquat Toxicol ; 214: 105236, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31260825

RESUMO

Otolith consisting largely of calcium carbonate, fibrous and proteins, is vital for maintaining body balance and/or hearing of fish. The formation of otolith involves Ca2+ transport and deposition. In the present study, we investigated the effects of Cd2+ on otoliths development by using zebrafish embryos as model. The results showed that exposure to Cd2+ inhibited the utricular and saccular otoliths growth, indicated by reduced lateral areas. Swimming speeds were reduced and a losing balance control was observed in Cd2+ exposed larvae. The genes related to Ca2+ transport (e.g. plasma membrane Ca2+-ATPase isoform 2, pmca2; Ca2+-ATPase isoform 2, atp2b1a) and regulation (e.g. parathyroid hormone ligand type-1, pth1; stanniocalcin isoform 1, stc1) were significantly downregulated. However, the adverse effects of Cd2+ on otoliths growth and swimming activity can be protected by supplementation of Ca2+ in exposure medium. Body burden of Cd2+ in larvae was reduced upon the supplement with Ca2+. The overall results suggest that exposure to Cd2+ can inhibit influx of Ca2+, leading to less deposition of CaCO3 for otolith growth, and finally result in impaired balance control and swimming activity in zebrafish larvae.


Assuntos
Comportamento Animal , Cádmio/toxicidade , Exposição Ambiental , Membrana dos Otólitos/crescimento & desenvolvimento , Peixe-Zebra/fisiologia , Animais , Comportamento Animal/efeitos dos fármacos , Cálcio/análise , Larva/efeitos dos fármacos , Atividade Motora/efeitos dos fármacos , Membrana dos Otólitos/efeitos dos fármacos , Fatores de Tempo , Testes de Toxicidade , Transcrição Genética/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
5.
Aquat Toxicol ; 214: 105240, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31319295

RESUMO

Fish has a strong resistance to microcystins (MCs), cyclic heptapeptide cyanotoxins, known as endocrine disrupting chemicals (EDCs) which are released during cyanobacterial blooms and many laboratory and field studies have found the hepatic recovery of fish from the MCs exposure. The aim of the present study was to investigate the recovery mechanisms of reproductive function of adult zebrafish (Danio rerio) from microcystin-LR (MC-LR) exposure. Therefore, adult female zebrafish were exposed to 0, 1 or 50 µg/L of MC-LR for 21days and transferred to MC free water for another 21 days to investigate the recovery. After MC-LR exposure, marked histological lesions in the gonads, decreased the percentage of mature oocytes, decreased number of spawned eggs, decreased fertilization and hatching rates were observed. MC-LR exposure increased the concentration of 17ß-estradiol (E2), testosterone (T) and vitellogenin (VTG) in female zebrafish. Some gene transcriptions of the hypothalamic-pituitary-gonad (HPG) axis significantly changed. The protein levels of 17ßhsd and cyp19a remarkably increased in the MC-LR exposure groups. However, our laboratory observation also indicates that zebrafish transferred from microcystin exposure to toxin-free water and reared for 21 days exhibited a nearly complete recovery of reproductive functions, including histological structure, increased the percentage of matured oocytes and spawned eggs, stable hormone levels, well-balanced transcriptional and translational levels. These results indicate that after MC-LR exposure, the reproductive impairments in zebrafish are also reversible likewise hepatic recovery seen by different studies in fish. Future studies should be conducted to explore a better understanding of the recovery mechanisms of fish from microcystins exposure.


Assuntos
Exposição Ambiental , Microcistinas/toxicidade , Reprodução/efeitos dos fármacos , Peixe-Zebra/fisiologia , Animais , Disruptores Endócrinos/toxicidade , Feminino , Hormônios/sangue , Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismo , Ovário/citologia , Ovário/efeitos dos fármacos , Ovário/fisiologia , Hipófise/efeitos dos fármacos , Hipófise/metabolismo , Transcrição Genética/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Peixe-Zebra/sangue , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
6.
Aquat Toxicol ; 213: 105219, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31195325

RESUMO

Nrf2 is a crucial transcription factor that regulates the expression of cytoprotective enzymes and controls cellular redox homeostasis. Both arsenic and fluoride are potent toxicants that are known to induce Nrf2. They are reported to coexist in many areas of the world leading to complex mixture effects in exposed organisms. The present study investigated the expression of Nrf2 and related xenobiotic metabolizing enzymes along with other stress markers such as histopathological alterations, catalase activity, reduced glutathione content and lipid peroxidation in zebrafish liver as a function of combined exposure to environmentally relevant concentrations of arsenic (37.87 µgL-1 or 5.05 × 10-7 M) and fluoride (6.8 mg L-1 or 3.57 × 10-4 M) for 60 days. The decrease in the total reduced glutathione level was evident in all treatment conditions. Hyperactivity of catalase along with conspicuous elevation in reactive oxygen species, malondialdehyde content and histo-architectural anomalies signified the presence of oxidative stress in the treatment groups. Nrf2 was seen to be induced at both transcriptional and translational levels in case of both individual and co-exposure. The same pattern was observed in case of its nuclear translocation also. From the results of qRT-PCR it was evident that at each time point co-exposure to arsenic and fluoride seemed to alter the gene expression of Cu/Zn Sod, Mn Sod, Gpx and Nqo1 just like their individual exposure but at a very low magnitude. In conclusion, this study demonstrates for the first time the differential expression and activity of Nrf2 and other stress response genes in the zebrafish liver following individual and combined exposure to arsenic and fluoride.


Assuntos
Arsênico/toxicidade , Fluoretos/toxicidade , Regulação da Expressão Gênica/efeitos dos fármacos , Fígado/enzimologia , Fígado/metabolismo , Fator 2 Relacionado a NF-E2/genética , Xenobióticos/metabolismo , Peixe-Zebra/metabolismo , Animais , Catalase/metabolismo , Glutationa/metabolismo , Glutationa Peroxidase/metabolismo , Fígado/efeitos dos fármacos , Fígado/patologia , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Poluentes Químicos da Água/toxicidade , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
7.
Neuron ; 103(4): 686-701.e8, 2019 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-31248729

RESUMO

The role of serotonin (5-HT) in sleep is controversial: early studies suggested a sleep-promoting role, but eventually the paradigm shifted toward a wake-promoting function for the serotonergic raphe. Here, we provide evidence from zebrafish and mice that the raphe are critical for the initiation and maintenance of sleep. In zebrafish, genetic ablation of 5-HT production by the raphe reduces sleep, sleep depth, and the homeostatic response to sleep deprivation. Pharmacological inhibition or ablation of the raphe reduces sleep, while optogenetic stimulation increases sleep. Similarly, in mice, ablation of the raphe increases wakefulness and impairs the homeostatic response to sleep deprivation, whereas tonic optogenetic stimulation at a rate similar to baseline activity induces sleep. Interestingly, burst optogenetic stimulation induces wakefulness in accordance with previously described burst activity of the raphe during arousing stimuli. These results indicate that the serotonergic system promotes sleep in both diurnal zebrafish and nocturnal rodents. VIDEO ABSTRACT.


Assuntos
Camundongos/fisiologia , Núcleos da Rafe/fisiologia , Serotonina/fisiologia , Sono/fisiologia , Peixe-Zebra/fisiologia , Animais , Nível de Alerta/genética , Nível de Alerta/fisiologia , Buspirona/farmacologia , Ritmo Circadiano/fisiologia , Fenclonina/farmacologia , Homeostase , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Optogenética , Quipazina/farmacologia , Neurônios Serotoninérgicos/efeitos dos fármacos , Neurônios Serotoninérgicos/fisiologia , Serotonina/biossíntese , Antagonistas da Serotonina/farmacologia , Agonistas do Receptor de Serotonina/farmacologia , Privação do Sono/genética , Privação do Sono/fisiopatologia , Triptofano Hidroxilase/deficiência , Triptofano Hidroxilase/genética , Vigília/genética , Vigília/fisiologia , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genética
8.
Mol Immunol ; 112: 206-214, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31176200

RESUMO

Neutrophil migration is essential for battling against infections but also drives chronic inflammation. Since primary neutrophils are terminally differentiated and not genetically tractable, leukemia cells such as HL-60 are differentiated into neutrophil-like cells to study mechanisms underlying neutrophil migration. However, constitutive overexpression or inhibition in this cell line does not allow the characterization of the genes that affect the differentiation process. Here we apply the tet-on system to induce the expression of a zebrafish microRNA, dre-miR-722, in differentiated HL-60. Overexpression of miR-722 reduced the mRNA level of genes in the chemotaxis and inflammation pathways, including Ras-Related C3 Botulinum Toxin Substrate 2 (RAC2). Consistently, polarization of the actin cytoskeleton, cell migration and generation of the reactive oxygen species are significantly inhibited upon induced miR-722 overexpression. Together, zebrafish miR-722 is a suppressor for migration and signaling in human neutrophil like cells.


Assuntos
Quimiotaxia/genética , MicroRNAs/genética , Neutrófilos/fisiologia , Peixe-Zebra/genética , Actinas/genética , Animais , Diferenciação Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/genética , Células HEK293 , Células HL-60 , Humanos , Inflamação/genética , Leucemia/genética , RNA Mensageiro/genética , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/genética , Proteínas de Peixe-Zebra/genética , Proteínas rac de Ligação ao GTP/genética
9.
PLoS Genet ; 15(6): e1008213, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31199790

RESUMO

The neural crest (NC) is a vertebrate-specific cell type that contributes to a wide range of different tissues across all three germ layers. The gene regulatory network (GRN) responsible for the formation of neural crest is conserved across vertebrates. Central to the induction of the NC GRN are AP-2 and SoxE transcription factors. NC induction robustness is ensured through the ability of some of these transcription factors to compensate loss of function of gene family members. However the gene regulatory events underlying compensation are poorly understood. We have used gene knockout and RNA sequencing strategies to dissect NC induction and compensation in zebrafish. We genetically ablate the NC using double mutants of tfap2a;tfap2c or remove specific subsets of the NC with sox10 and mitfa knockouts and characterise genome-wide gene expression levels across multiple time points. We find that compensation through a single wild-type allele of tfap2c is capable of maintaining early NC induction and differentiation in the absence of tfap2a function, but many target genes have abnormal expression levels and therefore show sensitivity to the reduced tfap2 dosage. This separation of morphological and molecular phenotypes identifies a core set of genes required for early NC development. We also identify the 15 somites stage as the peak of the molecular phenotype which strongly diminishes at 24 hpf even as the morphological phenotype becomes more apparent. Using gene knockouts, we associate previously uncharacterised genes with pigment cell development and establish a role for maternal Hippo signalling in melanocyte differentiation. This work extends and refines the NC GRN while also uncovering the transcriptional basis of genetic compensation via paralogues.


Assuntos
Desenvolvimento Embrionário/genética , Crista Neural/crescimento & desenvolvimento , Fatores de Transcrição SOXE/genética , Fator de Transcrição AP-2/genética , Proteínas de Peixe-Zebra/genética , Animais , Diferenciação Celular/genética , Linhagem da Célula/genética , Embrião não Mamífero , Regulação da Expressão Gênica no Desenvolvimento/genética , Redes Reguladoras de Genes/genética , Melanócitos/metabolismo , Fator de Transcrição Associado à Microftalmia/genética , Crista Neural/metabolismo , Pigmentação/genética , Proteínas Serina-Treonina Quinases/genética , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento
10.
Artigo em Inglês | MEDLINE | ID: mdl-31176867

RESUMO

In rice field eel (Monopterus albus), germ cell development in the developing gonad has been revealed in detail. However, it is unclear how primordial germ cells (PGCs) migrate to the somatic part of the gonad (genital ridge). This study visualized PGC migration by injecting a chimeric mRNA containing a fluorescent protein fused to the 3' untranslated region (3'UTR) of three different genes, nanos3 of zebrafish (Danio rerio) and dead end (dnd) and vasa of rice field eel. The mRNAs were injected either alone or in pairs into embryos at the one-cell stage. The results showed that mRNAs containing nanos3 and dnd 3'UTRs labeled PGCs over a wider time frame than those containing vasa 3'UTR, suggesting that nanos3 and dnd 3'UTRs are suitable for visualizing PGCs in rice field eel. Using this direct visualization method, the normal migration route of PGCs was observed from the 50%-epiboly stage to hatching stage for the first time, and the ectopic PGCs were also visualized during this period in rice field eel. These findings extend our knowledge of germ cell development, and lay a foundation for further research on the relationship between PGCs and sex differentiation, and on incubation conditions for embryos in rice field eel.


Assuntos
Células Germinativas/metabolismo , Smegmamorpha/embriologia , Regiões 3' não Traduzidas , Sequência de Aminoácidos , Animais , Movimento Celular/genética , Movimento Celular/fisiologia , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Feminino , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Células Germinativas/citologia , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , RNA/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Smegmamorpha/genética , Smegmamorpha/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
11.
Chemosphere ; 228: 649-655, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31063912

RESUMO

Circadian rhythms are fundamental to behavior and physiology of organisms. Flutolanil as a fungicide is toxic to zebrafish embryos. The aims of this study were to determine whether flutolanil would influence circadian rhythms of zebrafish and the mechanism involved. Zebrafish embryos were exposed to flutolanil (0, 0.125, 0.5 and 2 mg/L) for 4 days. Here we report that flutolanil increased the melatonin levels of zebrafish. The mRNA levels of genes related to circadian rhythms were significantly altered. The clock level was significantly increased, but the content of cry1 showed no apparent changes. Moreover, our findings that the level of GH was significantly decreased were consistent with the abnormal development of zebrafish embryos. The expression levels of genes related to development, behavior and reproduction were significantly altered by flutolanil. These results indicate that flutolanil disturbed circadian rhythms of zebrafish primarily by affecting the positive elements, which were at least in partial responsible for abnormal development and behavior of zebrafish. And we speculate that flutolanil is toxic to zebrafish embryos at least in part via dysregulation of circadian rhythms involving clock.


Assuntos
Anilidas/toxicidade , Ritmo Circadiano/efeitos dos fármacos , Melatonina/metabolismo , Peixe-Zebra/embriologia , Animais , Comportamento Animal/efeitos dos fármacos , Proteínas CLOCK/genética , Ritmo Circadiano/genética , Criptocromos/genética , Ecotoxicologia/métodos , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/fisiologia , Fungicidas Industriais/toxicidade , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Peixe-Zebra/fisiologia , Proteínas de Peixe-Zebra/genética
12.
Chemosphere ; 229: 169-180, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31078031

RESUMO

Although silver nanoparticles (AgNPs) are used in various commercial products, the biological effects of AgNPs on fish embryogenesis and the underlying molecular mechanisms are still poorly understood. In this study, both touch responses and neuron membrane potential were found to be abnormal in AgNPs-stressed embryos. Moreover, neurogenesis genes were unveiled to be down-regulated and were enriched in ligand-gated ion channel activity, dopamine receptor signaling pathway, etc. in AgNPs-stressed embryos by microarray assays. Additionally, the down-regulated expression of otpa/sncgb - gad1b/gad2 dopaminergic neurotransmitter genes, robo2 - vim and glrbb synaptic transmission genes, and motor neuron genes isl1 &isl2a was further identified in both AgNPs- and Ag+-stressed embryos by qPCR, whole-mount in situ hybridization (WISH), and by using specific promoter-derived GFP fluorescence transgenic zebrafish. Moreover, the reduced expression of gad1b, gad2, and isl1 could be recovered by adding Ag+ chelating compound l-cysteine in AgNPs stressed embryos. Our results reveal for the first time that it is through damaging the formation of neural circuits, including dopaminergic neurotransmitter, synaptic transmission, and motor activities, that AgNPs induce abnormal electrical membrane properties, leading to dysfunctional touch responses and locomotor escape responses mostly via their released Ag+ during embryogenesis.


Assuntos
Embrião não Mamífero/efeitos dos fármacos , Nanopartículas Metálicas/toxicidade , Fenômenos Fisiológicos do Sistema Nervoso/efeitos dos fármacos , Prata/química , Prata/toxicidade , Tato/efeitos dos fármacos , Peixe-Zebra/embriologia , Animais , Embrião não Mamífero/fisiologia , Tato/fisiologia , Peixe-Zebra/metabolismo , Peixe-Zebra/fisiologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
13.
Int J Mol Sci ; 20(9)2019 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-31052497

RESUMO

The purpose of the present study is to evaluate the effect of rice bran ash mineral extract (RBM) on pigmentation in zebrafish (Danio rerio). Melanin has the ability to block ultraviolet (UV) radiation and scavenge free oxygen radicals, thus protecting the skin from their harmful effects. Agents that increase melanin synthesis in melanocytes may reduce the risk of photodamage and skin cancer. The present study investigates the effect of RBM on pigmentation in zebrafish and the underlying mechanism. RBM was found to significantly increase the expression of microphthalmia-associated transcription factor (MITF), a key transcription factor involved in melanin production. RBM also suppressed the phosphorylation of extracellular signal-regulated kinase (ERK), which negatively regulates zebrafish pigmentation. Together, these results suggest that RBM promotes melanin biosynthesis in zebrafish.


Assuntos
Oryza/química , Pigmentação/efeitos dos fármacos , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Peixe-Zebra/fisiologia , Animais , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Melaninas/metabolismo , Fator de Transcrição Associado à Microftalmia/genética , Fator de Transcrição Associado à Microftalmia/metabolismo , Fosforilação/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
14.
J Toxicol Sci ; 44(5): 347-356, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31068540

RESUMO

Increasing use of zebrafish in biomedical, toxicological and developmental studies requires explicit knowledge of cytochrome P450 (CYP), given the central role of CYP in oxidative biotransformation of xenobiotics and many regulatory molecules. A full complement of CYP genes in zebrafish and their transcript expression during early development have already been examined. Here we established a comprehensive picture of CYP gene expression in the adult zebrafish liver using a RNA-seq technique. Transcriptional profiling of a full complement of CYP genes revealed that CYP2AD2, CYP3A65, CYP1A, CYP2P9 and CYP2Y3 are major CYP genes expressed in the adult zebrafish liver in both sexes. Quantitative real-time RT-PCR analysis for selected CYP genes further supported our RNA-seq data. There were significant sex differences in the transcript levels for CYP1A, CYP1B1, CYP1D1 and CYP2N13, with males having higher expression levels than those in females in all cases. A similar feature of gender-specific expression was observed for CYP2AD2 and CYP2P9, suggesting sex-specific regulation of constitutive expression of some CYP genes in the adult zebrafish liver. The present study revealed several "orphan" CYP genes as dominant isozymes at transcript levels in the adult zebrafish liver, implying crucial roles of these CYP genes in liver physiology and drug metabolism. The current results establish a foundation for studies with zebrafish in drug discovery and toxicology.


Assuntos
Sistema Enzimático do Citocromo P-450/genética , Fígado/metabolismo , Proteínas de Peixe-Zebra/genética , Animais , Feminino , Perfilação da Expressão Gênica , Masculino , Peixe-Zebra
15.
Nat Cell Biol ; 21(6): 721-730, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31110287

RESUMO

Wnt signalling drives many processes in development, homeostasis and disease; however, the role and mechanism of individual ligand-receptor (Wnt-Frizzled (Fzd)) interactions in specific biological processes remain poorly understood. Wnt9a is specifically required for the amplification of blood progenitor cells during development. Using genetic studies in zebrafish and human embryonic stem cells, paired with in vitro cell biology and biochemistry, we determined that Wnt9a signals specifically through Fzd9b to elicit ß-catenin-dependent Wnt signalling that regulates haematopoietic stem and progenitor cell emergence. We demonstrate that the epidermal growth factor receptor (EGFR) is required as a cofactor for Wnt9a-Fzd9b signalling. EGFR-mediated phosphorylation of one tyrosine residue on the Fzd9b intracellular tail in response to Wnt9a promotes internalization of the Wnt9a-Fzd9b-LRP signalosome and subsequent signal transduction. These findings provide mechanistic insights for specific Wnt-Fzd signals, which will be crucial for specific therapeutic targeting and regenerative medicine.


Assuntos
Células-Tronco Hematopoéticas/citologia , Receptores de Neurotransmissores/genética , Proteínas Wnt/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Receptores ErbB/genética , Humanos , Fosforilação , Via de Sinalização Wnt , Peixe-Zebra/crescimento & desenvolvimento , beta Catenina/genética
16.
BMC Genomics ; 20(1): 341, 2019 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-31060508

RESUMO

BACKGROUND: Elevated water temperature, as is expected through climate change, leads to masculinization in fish species with sexual plasticity, resulting in changes in population dynamics. These changes are one important ecological consequence, contributing to the risk of extinction in small and inbred fish populations under natural conditions, due to male-biased sex ratio. Here we investigated the effect of elevated water temperature during embryogenesis on sex ratio and sex-biased gene expression profiles between two different tissues, namely gonad and caudal fin of adult zebrafish males and females, to gain new insights into the molecular mechanisms underlying sex determination (SD) and colour patterning related to sexual attractiveness. RESULTS: Our study demonstrated sex ratio imbalances with 25.5% more males under high-temperature condition, resulting from gonadal masculinization. The result of transcriptome analysis showed a significantly upregulated expression of male SD genes (e.g. dmrt1, amh, cyp11c1 and sept8b) and downregulation of female SD genes (e.g. zp2.1, vtg1, cyp19a1a and bmp15) in male gonads compared to female gonads. Contrary to expectations, we found highly differential expression of colour pattern (CP) genes in the gonads, suggesting the 'neofunctionalisation' of those genes in the zebrafish reproduction system. However, in the caudal fin, no differential expression of CP genes was identified, suggesting the observed differences in colouration between males and females in adult fish may be due to post-transcriptional regulation of key enzymes involved in pigment synthesis and distribution. CONCLUSIONS: Our study demonstrates male-biased sex ratio under high temperature condition and support a polygenic SD (PSD) system in laboratory zebrafish. We identify a subset of pathways (tight junction, gap junction and apoptosis), enriched for SD and CP genes, which appear to be co-regulated in the same pathway, providing evidence for involvement of those genes in the regulation of phenotypic sexual dimorphism in zebrafish.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Gônadas/metabolismo , Diferenciação Sexual , Razão de Masculinidade , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/genética , Animais , Cor , Feminino , Temperatura Alta , Masculino , Maturidade Sexual , Transcriptoma , Peixe-Zebra/fisiologia , Proteínas de Peixe-Zebra/genética
17.
Int J Mol Sci ; 20(10)2019 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-31130675

RESUMO

Site-specific incorporation of un-natural amino acids (UNAA) is a powerful approach to engineer and understand protein function. Site-specific incorporation of UNAAs is achieved through repurposing the amber codon (UAG) as a sense codon for the UNAA, using a tRNACUA that base pairs with an UAG codon in the mRNA and an orthogonal amino-acyl tRNA synthetase (aaRS) that charges the tRNACUA with the UNAA. Here, we report an expansion of the zebrafish genetic code to incorporate the UNAAs, azido-lysine (AzK), bicyclononyne-lysine (BCNK), and diazirine-lysine (AbK) into green fluorescent protein (GFP) and glutathione-s-transferase (GST). We also present proteomic evidence for UNAA incorporation into GFP. Our work sets the stage for the use of AzK, BCNK, and AbK introduction into proteins as a means to investigate and engineer their function in zebrafish.


Assuntos
Lisina/análogos & derivados , Engenharia de Proteínas/métodos , Peixe-Zebra/genética , Animais , Códon de Terminação/genética , Código Genético , Glutationa Transferase/genética , Proteínas de Fluorescência Verde/genética , Lisina/genética , Proteínas de Peixe-Zebra/genética
18.
Aquat Toxicol ; 212: 88-97, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31077970

RESUMO

Ionocytes are specialized cells in the epidermis of embryonic zebrafish (Danio rerio) that play important roles in ion homeostasis and have functional similarities to mammalian renal cells. Here, we examined whether these cells might also share another functional similarity with renal cells, which is the presence of efflux transporter activities useful for elimination of toxic small molecules. Xenobiotic transporters (XTs), including the ATP-Binding Cassette (ABC) family, are a major defense mechanism against diffusible toxic molecules in aquatic embryos, including zebrafish, but their activity in the ionocytes has not previously been studied. Using fluorescent small molecule substrates of XT, we observed that specific populations of ionocytes uptake and efflux fluorescent small molecules in a manner consistent with active transport. We specifically identified a P-gp/ABCB1 inhibitor-sensitive efflux activity in the H+-ATPase-rich (HR) ionocytes, and show that these cells exhibit enriched expression of the ABCB gene, abcb5. The results extend our understanding of the functional significance of zebrafish ionocytes and indicate that these cells could play an important role in protection of the fish embryo from harmful small molecules.


Assuntos
Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Xenobióticos/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Animais , Ânions , Transporte Biológico , Epiderme/efeitos dos fármacos , Corantes Fluorescentes/metabolismo , Mitocôndrias/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Proteínas de Peixe-Zebra/genética
19.
Int J Mol Sci ; 20(8)2019 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-31014006

RESUMO

Pancreatic endocrine cells expressing the ghrelin gene and producing the ghrelin hormone were first identified in 2002. These cells, named ε cells, were recognized as the fifth type of endocrine cells. Differentiation of ε cells is induced by various transcription factors, including Nk2 homeobox 2, paired box proteins Pax-4 and Pax6, and the aristaless-related homeobox. Ghrelin is generally considered to be a "hunger hormone" that stimulates the appetite and is produced mainly by the stomach. Although the population of ε cells is small in adults, they play important roles in regulating other endocrine cells, especially ß cells, by releasing ghrelin. However, the roles of ghrelin in ß cells are complex. Ghrelin contributes to increased blood glucose levels by suppressing insulin release from ß cells and is also involved in the growth and proliferation of ß cells and the prevention of ß cell apoptosis. Despite increasing evidence and clarification of the mechanisms of ε cells over the last 20 years, many questions remain to be answered. In this review, we present the current evidence for the participation of ε cells in differentiation and clarify their characteristics by focusing on the roles of ghrelin.


Assuntos
Ilhotas Pancreáticas/metabolismo , Animais , Grelina/genética , Grelina/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
20.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 35(2): 134-139, 2019 Feb.
Artigo em Chinês | MEDLINE | ID: mdl-30975277

RESUMO

Objective To explore the relationship between the drug resistance gene ATP binding cassette sub-family B member 4 (abcb4) in zebrafish and Wnt/ß-catenin signaling pathway. Methods Wild-type zebrafish and transgenic zebrafish were set in the blank control group, doxorubicin treatment group, vinblastine treatment group, gefitinib treatment group, doxorubicin combined with gefitinib treatment group, and vinblastine combined with gefitinib treatment group. The 100 embryos of each group were treated with drugs until the 5th day, and 50 zebrafish juveniles were tested on the 5th day in each group. The drug resistance of wild-type zebrafish was tested by rhodamine 123 experiment. The fluorescence intensity of transgenic zebrafish was tested by microplate reader, and the fluorescence distribution was tested by living cell workstation. The protein levels of transgenic zebrafish ß-catenin, GSK-3ß, ABCB4 and enhanced green fluorescent protein (EGFP) were tested by Western blot analysis. Results Rhodamine 123 experiments proved that there was drug resistance in zebrafish when treated with doxorubicin and vinblastine. Compared with the blank group, the EGFP fluorescence intensity increased in the transgenic zebrafish when treated with doxorubicin and vinblastine. Western blot assay showed the accumulation of ß-catenin accompanied by the increase of EGFP and ABCB4 proteins in the transgenic zebrafish exposed to adriamycin and vincristine. Conclusion The Wnt/ß-catenin signaling pathway in zebrafish is involved in the activation and drug resistance of zebrafish abcb4 gene.


Assuntos
Resistência a Medicamentos , Via de Sinalização Wnt , Proteínas de Peixe-Zebra , Peixe-Zebra , beta Catenina , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Animais Geneticamente Modificados , Antineoplásicos/farmacologia , Resistência a Medicamentos/genética , Embrião não Mamífero/efeitos dos fármacos , Via de Sinalização Wnt/fisiologia , Proteínas de Peixe-Zebra/genética , beta Catenina/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA