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1.
Gene ; 751: 144761, 2020 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-32407768

RESUMO

The ubiquitin specific peptidase (USP) family is involved in many life processes, of which antiviral is also an important basic function. One of the more important ways is to activate interferon. In this study, we reported the antiviral function of the ubiquitin specific peptidase 5(USP5) gene in zebrafish. Evolutionary and comparative protein sequence analysis of the USP5 was performed. The localization of USP5 in FHM cells cytoplasm was determined. Overexpression of USP5 significantly evoked higher expression of mRNA that encode IFNφ1 and ISGs, the promoteractivities of IFNφ1 and IFNstimulated response element (ISRE) were augmented likewise. USP5 was also able to enhance the expression of RIG-I and activate higher levels of IFNφ1 stimulated by Poly (I: C). Viral infection and interference tests demonstrated that USP5 inhibited the replication of SVCV in vitro. In summary, this study reveals that USP5 is able to activate higher levels of interferon by increasing RIG-I protein levels, and thus implement antivirus functions.


Assuntos
Proteases Específicas de Ubiquitina/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Sequência de Aminoácidos , Animais , Células Cultivadas , Interferons/biossíntese , Interferons/genética , Rhabdoviridae/fisiologia , Alinhamento de Sequência , Proteases Específicas de Ubiquitina/química , Proteases Específicas de Ubiquitina/genética , Proteases Específicas de Ubiquitina/metabolismo , Replicação Viral , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Peixe-Zebra/virologia , Proteínas de Peixe-Zebra/biossíntese , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
2.
PLoS One ; 15(3): e0225917, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32142555

RESUMO

Nutritional Programming (NP) has been shown to counteract the negative effects of dietary plant protein (PP) by introducing PP at an early age towards enhancement of PP utilization during later life stages. This study explored the effect of NP and its induction time on growth, expression of appetite-stimulating hormones, and any morphological changes in the gut possibly responsible for improved dietary PP utilization. At 3 days post-hatch (dph) zebrafish were distributed into 12 (3 L) tanks, 100 larvae per tank. This study included four groups: 1) The control (NP-FM) group received fishmeal (FM)-based diet from 13-36 dph and was challenged with PP-based diet during 36-66 dph; 2) The NP-PP group received NP with dietary PP in larval stage via live food enrichment during 3-13 dph followed by FM diet during 13-36 dph and PP diet during 36-66 dph; 3) The T-NP group received NP between 13-23 dph through PP diet followed by FM diet during 23-36 dph and PP diet during 36-66 dph; and 4) The PP group received PP diet from 13-66 dph. During the PP challenge the T-NP group achieved the highest weight gain compared to control and PP. Ghrelin expression in the brain was higher in T-NP compared to NP-FM and NP-PP, while in the gut it was reduced in both NP-PP and T-NP groups. Cholecystokinin expression showed an opposite trend to ghrelin. The brain neuropeptide Y expression was lower in NP-PP compared to PP but not different with NP-FM and T-NP groups. The highest villus length to width ratio in the middle intestine was found in T-NP compared to all other groups. The study suggests that NP induced during juvenile stages improves zebrafish growth and affects digestive hormone regulation and morphology of the intestinal lining-possible mechanisms behind the improved PP utilization in pre-adult zebrafish stages.


Assuntos
Ração Animal , Encéfalo/metabolismo , Colecistocinina/biossíntese , Grelina/biossíntese , Proteínas de Vegetais Comestíveis/farmacologia , Proteínas de Peixe-Zebra/biossíntese , Peixe-Zebra/metabolismo , Animais
3.
Artigo em Inglês | MEDLINE | ID: mdl-31669374

RESUMO

Muscle-specific RING-finger proteins (MuRFs) are E3 ubiquitin ligases that play important roles in protein quality control in skeletal and cardiac muscles. Here we characterized murf gene expression and protein localization in zebrafish embryos. We found that the zebrafish genome contains six murf genes, including murf1a, murf1b, murf2a, murf2b, murf3 and a murf2-like gene that are specifically expressed in skeletal and cardiac muscles of zebrafish embryos. To analyze the subcellular localization, we generated transgenic zebrafish models expressing MurF1a-GFP or MuRF2a-GFP fusion proteins. MuRF1a-GFP and MuRF2a-GFP showed distinct patterns of subcellular localization. MuRF1a-GFP displayed a striated pattern of localization in myofibers, whereas MuRF2a-GFP mainly exhibited a random pattern of punctate distribution. The MuRF1a-GFP signal appeared as small dots aligned along the M-lines of the sarcomeres in skeletal myofibers. To determine whether knockdown of smyd1b or hsp90α1 that increased myosin protein degradation could alter murf gene expression or MuRF protein localization, we knocked down smyd1b or hsp90α1 in wild type, Tg(ef1a:MurF1a-GFP) and Tg(ef1a:MuRF2a-GFP) transgenic zebrafish embryos. Knockdown of smyd1b or hsp90α1 had no effect on murf gene expression. However, the sarcomeric distribution of MuRF1a-GFP was abolished in the knockdown embryos. This was accompanied by an increased random punctate distribution of MuRF1a-GFP in muscle cells of zebrafish embryos. Collectively, these studies demonstrate that MuRFs are specifically expressed in developing muscles of zebrafish embryos. The M-line localization MuRF1a is altered by sarcomere disruption in smyd1b or hsp90α1 knockdown embryos.


Assuntos
Animais Geneticamente Modificados , Embrião não Mamífero/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Choque Térmico HSP90 , Histona-Lisina N-Metiltransferase , Modelos Biológicos , Proteínas Recombinantes de Fusão , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Animais Geneticamente Modificados/embriologia , Animais Geneticamente Modificados/genética , Proteínas de Choque Térmico HSP90/biossíntese , Proteínas de Choque Térmico HSP90/genética , Histona-Lisina N-Metiltransferase/biossíntese , Histona-Lisina N-Metiltransferase/genética , Músculo Esquelético/embriologia , Proteínas Recombinantes de Fusão/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/biossíntese , Proteínas de Peixe-Zebra/genética
4.
Chemosphere ; 238: 124589, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31437630

RESUMO

Vitellogenin (VTG), a well-established biomarker for the diagnosis of endocrine activity in fish, is used in multiple OECD test guidelines (TG) to identify activities of chemicals on hormonal pathways. However, the synthesis of VTG may not only be modified by typical endocrine-related pathways, but also through non-endocrine-mediated processes. In particular, hepatotoxicity, i.e. toxicant-induced impairment of liver structure and function, might influence VTG as a biomarker, since VTG is synthesized in hepatocytes. An intimate understanding of the interplay between endocrine-related and non-endocrine-related pathways influencing VTG production is crucial for the avoidance of erroneous diagnoses in hazard assessment for regulatory purposes of chemical compounds. In order to investigate whether hepatotoxicity may interfere with hepatic VTG synthesis, adult zebrafish (Danio rerio) were exposed to three well-known hepatotoxicants, acetaminophen, isoniazid and acetylsalicylic acid, according to OECD TG 230. Various hepatotoxicity- and endocrine system-related endpoints were recorded: mRNA expression of selected endocrine- and hepatotoxicity-related marker genes in the liver; VTG levels in head/tail homogenates; and liver histopathology. All three test compounds induced significant, but mild single cell necrosis of hepatocytes and transcriptional changes of hepatotoxicity-related marker genes, thus confirming hepatotoxic effects. A positive correlation between hepatotoxicity and reduced hepatic VTG synthesis was not observed, with the single exception of a weak increase in female zebrafish exposed to APAP. This suggests that - in studies conducted according to OECD TG 229 or 230 - it is unlikely that hepatotoxic chemicals will interfere with the hepatic capacity for VTG synthesis.


Assuntos
Acetaminofen/toxicidade , Aspirina/toxicidade , Hepatócitos/metabolismo , Isoniazida/toxicidade , Vitelogeninas/biossíntese , Poluentes Químicos da Água/toxicidade , Peixe-Zebra/metabolismo , Animais , Doença Hepática Induzida por Substâncias e Drogas , Sistema Endócrino/efeitos dos fármacos , Feminino , Hepatócitos/efeitos dos fármacos , Masculino , Proteínas de Peixe-Zebra/biossíntese
5.
Chemosphere ; 238: 124584, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31470313

RESUMO

Exposure to endocrine disrupting chemicals has been suggested to contribute to the ongoing globally increasing obesity trend. The complex chemical mixtures that humans and wildlife are exposed to include a number of compounds that may have obesogenic properties. In this study we examined a mixture consisting of phthalate-monoesters, triclosan, and perfluorinated compounds. The mixture was designed within the EDC-MixRisk project based on serum levels of the compounds in pregnant women of a Swedish mother-child cohort. The compounds were negatively associated with birth weight of the children. We assessed whether developmental exposure to this mixture in combination with a calorie-rich diet affected metabolic rate, blood lipids, adipogenesis and lipid storage, and the whole-body level of neutral lipids in zebrafish (Danio rerio). Wildtype zebrafish were exposed to the mixture from 3 h post fertilization to 5, 14 or 17 days post fertilization (dpf) at water concentrations corresponding to 1, 10, 20, or 100 times the geometrical mean of the serum concentration (hsc) in the women. Exposure to the mixture at 20 times hsc lowered metabolic rate at 2-5 dpf, and increased the number of adipocytes and the amount of visceral adipose tissue at 14 and 17 dpf respectively. Also, mRNA expression of fatty acid binding protein 11a was increased at 17 dpf by 10 and 20 times hsc of the mixture. This study shows that a human-relevant mixture of environmental pollutants affects metabolic rate, adipogenesis and lipid storage in young zebrafish fed a calorie-rich diet, thus demonstrating its potential to disrupt metabolism.


Assuntos
Adipogenia/efeitos dos fármacos , Metabolismo Basal/efeitos dos fármacos , Disruptores Endócrinos/toxicidade , Poluentes Químicos da Água/toxicidade , Peixe-Zebra/metabolismo , Animais , Peso ao Nascer/efeitos dos fármacos , Proteínas de Ligação a Ácido Graxo/biossíntese , Proteínas de Ligação a Ácido Graxo/genética , Feminino , Humanos , Hidrocarbonetos Fluorados/toxicidade , Ácidos Ftálicos/toxicidade , Gravidez , Triclosan/toxicidade , Proteínas de Peixe-Zebra/biossíntese , Proteínas de Peixe-Zebra/genética
6.
Elife ; 82019 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-31829936

RESUMO

Tcf7l2 mediates Wnt/ß-Catenin signalling during development and is implicated in cancer and type-2 diabetes. The mechanisms by which Tcf7l2 and Wnt/ß-Catenin signalling elicit such a diversity of biological outcomes are poorly understood. Here, we study the function of zebrafish tcf7l2alternative splice variants and show that only variants that include exon five or an analogous human tcf7l2 variant can effectively provide compensatory repressor function to restore eye formation in embryos lacking tcf7l1a/tcf7l1b function. Knockdown of exon five specific tcf7l2 variants in tcf7l1a mutants also compromises eye formation, and these variants can effectively repress Wnt pathway activity in reporter assays using Wnt target gene promoters. We show that the repressive activities of exon5-coded variants are likely explained by their interaction with Tle co-repressors. Furthermore, phosphorylated residues in Tcf7l2 coded exon5 facilitate repressor activity. Our studies suggest that developmentally regulated splicing of tcf7l2 can influence the transcriptional output of the Wnt pathway.


Assuntos
Olho/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Isoformas de Proteínas/biossíntese , Processamento de RNA , Proteína 2 Semelhante ao Fator 7 de Transcrição/biossíntese , Transcrição Genética , Proteínas de Peixe-Zebra/biossíntese , Animais , Células HEK293 , Humanos , Isoformas de Proteínas/genética , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , Proteína 2 Semelhante ao Fator 7 de Transcrição/metabolismo , Via de Sinalização Wnt , Peixe-Zebra , Proteínas de Peixe-Zebra/genética
7.
Artigo em Inglês | MEDLINE | ID: mdl-31499217

RESUMO

Two distinct nonapeptide systems, vasotocin- and oxytocin-related peptides, evolved in vertebrates. Their role in male zebrafish reproduction has not been formally investigated. We hypothesized that the teleost nonapeptides vasotocin and isotocin stimulate male zebrafish reproductive physiology and success by affecting central neuronal and/or peripheral endocrine pathways. Pharmacological inhibition experiments revealed that both vasotocin and isotocin contribute significantly to male reproductive success, which in the case of vasotocin correlated significantly with indices of male courtship behavior. Interestingly, co-administration of vasotocin and isotocin antagonists completely abolished male reproductive success without affecting male courtship behavior and endocrine indices, possibly linked to a synergistic action of nonapeptides on male pheromone release. To further probe the nonapeptides' role in male zebrafish reproduction, we subsequently tested whether male zebrafish nonapeptide systems were acutely activated by the female releaser pheromone PGF2α, a strong chemoattractant and important reproductive cue in males which stimulates courtship behavior. Male zebrafish attracted to PGF2α in a choice assay exhibited acute increases in neuronal activation marker p-ERK immunoreactivity in the ventral glomerulus of the olfactory bulb and the preoptic area, however no co-localization with isotocin was observed. Conversely, PGF2α time-dependently stimulated whole brain isotocin mRNA abundance, suggesting secondary longer-term effects of PGF2α exposure on the central isotocinergic system. While the current lack of vasotocin-specific antibodies for zebrafish does not allow to probe acute activation of vasotocinergic neurons, whole brain vasotocin mRNA was not significantly affected by PGF2α exposure. Together, our results identify a role for nonapeptides in male zebrafish reproductive physiology and success.


Assuntos
Encéfalo/metabolismo , Ocitocina/análogos & derivados , Reprodução/fisiologia , Vasotocina/biossíntese , Proteínas de Peixe-Zebra/biossíntese , Peixe-Zebra/metabolismo , Animais , Masculino , Neurônios/metabolismo , Ocitocina/biossíntese , Ocitocina/genética , Vasotocina/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
8.
Biomed Res Int ; 2019: 4508048, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31428635

RESUMO

The 6-O-endosulfatases (sulfs) are important enzymatic components involved in the regulation of heparan sulfate by altering the sulfatation pattern. Specifically in the kidney, sulfs have been implicated in the glomerular podocyte-endothelial cell crosstalk and in the preservation of the glomerular filtration barrier (GFB) in different mouse models. Since it has been shown that in zebrafish larvae, Sulf1, Sulf2a, and Sulf2b are expressed in the pronephric kidney we set out to establish if a reduction in sulf expression leads to GFB dysfunction. Here, we show that a reduced sulf expression following morpholino (MO) induced knockdown in zebrafish larvae promotes damage to the GFB leading to renal plasma protein loss from the circulation. Moreover, a combined knockdown of Sulf1, Sulf2a, and Sulf2b is associated with severe morphologic changes including narrowing of the fenestration between glomerular endothelial cells as well as thickening of the glomerular basement membrane and podocyte foot process effacement, suggesting that glomerular damage is an underlying cause of the circulatory protein loss observed after MO injection. Additionally, we show that a decrease in sulf expression reduces the bioavailability of VegfA in the glomerulus of the pronephros, which may contribute to the structural changes observed in the glomeruli of morphant fish. Furthermore, consistent with previous results, knockdown of the sulfs is associated with arteriovenous malformations in particular in the tail region of the larvae. Overall, taken together our results suggest that 6-O-endosulfatases are important in the preservation of GFB integrity and a reduction in their expression levels induces phenotypic changes that are indicative of renal protein loss.


Assuntos
Membrana Basal Glomerular/embriologia , Podócitos/enzimologia , Sulfatases/biossíntese , Proteínas de Peixe-Zebra/biossíntese , Peixe-Zebra/embriologia , Animais , Células Endoteliais/enzimologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Morfolinos/farmacologia , Sulfatases/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
9.
PLoS One ; 14(5): e0216370, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31048899

RESUMO

Actinotrichia are the first exoskeletal elements formed during zebrafish fin development. These rigid fibrils serve as skeletal support for the fin fold and as substrates for mesenchymal cell migration. In the adult intact fins, actinotrichia are restricted to the distal domain of the fin. Following fin amputation, actinotrichia also reform during regeneration. The actinodin gene family codes for structural proteins of actinotrichia. We have previously identified cis-acting regulatory elements in a 2kb genomic region upstream of the first exon of actinodin1, termed 2P, required for tissue-specific expression in the fin fold ectoderm and mesenchyme during embryonic development. Indeed, 2P contains an ectodermal enhancer in a 150bp region named epi. Deletion of epi from 2P results in loss of ectodermal-specific activity. In the present study, we sought to further characterize the activity of these regulatory sequences throughout fin development and during adult fin regeneration. Using a reporter transgenic approach, we show that a site within the epi region, termed epi3, contains an early mesenchymal-specific repressor. We also show that the larval fin fold ectodermal enhancer within epi3 remains functional in the basal epithelial layer during fin regeneration. We show that the first non-coding exon and first intron of actinodin1 contains a transcriptional enhancer and an alternative promoter that are necessary for the persistence of reporter expression reminiscent of actinodin1 expression during adulthood. Altogether, we have identified cis-acting regulatory elements that are required for tissue-specific expression as well as full recapitulation of actinodin1 expression during adulthood. Furthermore, the characterization of these elements provides us with useful molecular tools for the enhancement of transgene expression in adulthood.


Assuntos
Nadadeiras de Animais/fisiologia , Embrião não Mamífero/embriologia , Desenvolvimento Embrionário/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Regeneração/fisiologia , Proteínas de Peixe-Zebra/biossíntese , Peixe-Zebra/embriologia , Animais , Embrião não Mamífero/citologia , Elementos Facilitadores Genéticos/fisiologia , Éxons/fisiologia , Íntrons/fisiologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
10.
Blood Adv ; 3(8): 1244-1254, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30987969

RESUMO

Erythropoiesis is the process by which new red blood cells (RBCs) are formed and defects in this process can lead to anemia or thalassemia. The GATA1 transcription factor is an established mediator of RBC development. However, the upstream mechanisms that regulate the expression of GATA1 are not completely characterized. Cholesterol is 1 potential upstream mediator of GATA1 expression because previously published studies suggest that defects in cholesterol synthesis disrupt RBC differentiation. Here we characterize RBC development in a zebrafish harboring a single missense mutation in the hmgcs1 gene (Vu57 allele). hmgcs1 encodes the first enzyme in the cholesterol synthesis pathway and mutation of hmgcs1 inhibits cholesterol synthesis. We analyzed the number of RBCs in hmgcs1 mutants and their wild-type siblings. Mutation of hmgcs1 resulted in a decrease in the number of mature RBCs, which coincides with reduced gata1a expression. We combined these experiments with pharmacological inhibition and confirmed that cholesterol and isoprenoid synthesis are essential for RBC differentiation, but that gata1a expression is isoprenoid dependent. Collectively, our results reveal 2 novel upstream regulators of RBC development and suggest that appropriate cholesterol homeostasis is critical for primitive erythropoiesis.


Assuntos
Diferenciação Celular/genética , Eritrócitos/enzimologia , Eritropoese/genética , Hidroximetilglutaril-CoA Sintase , Mutação de Sentido Incorreto , Terpenos/metabolismo , Peixe-Zebra , Substituição de Aminoácidos , Animais , Colesterol/biossíntese , Colesterol/genética , Fator de Transcrição GATA1/biossíntese , Fator de Transcrição GATA1/genética , Regulação da Expressão Gênica , Hidroximetilglutaril-CoA Sintase/genética , Hidroximetilglutaril-CoA Sintase/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/biossíntese , Proteínas de Peixe-Zebra/genética
11.
Artigo em Inglês | MEDLINE | ID: mdl-30826460

RESUMO

Sensitization of motor activity is a behavioural test to evaluate the effects of psychostimulants. Conditioned place preference (CPP) is an associative learning procedure to examine the rewarding properties of drugs. We aimed to assess whether motor sensitization to drugs of abuse can make zebrafish more vulnerable to establishing drug-induced CPP. We first evaluated sensitization of locomotor activity of zebrafish to repeated administrations of nicotine and cocaine during 5 days and after 5 days of withdrawal. After withdrawal, when zebrafish were re-exposed to the same dose of nicotine or cocaine locomotor activity was increased by 103% and 166%, respectively. Different groups of zebrafish were sensitized to nicotine or cocaine and trained on a nicotine-CPP task the day after withdrawal. The nicotine dose selected for sensitization was not effective for developing CPP in naïve zebrafish whereas it elicited CPP in zebrafish that were previously sensitized to nicotine or cocaine. Levels of nicotinic acetylcholine receptor ß2, α6 and α7 subunit, Pitx3, and tyrosine hydroxylase 1 (TH1) mRNAs were increased in the brain of nicotine- and cocaine-sensitized zebrafish. Nicotine-CPP performed with drug-sensitized zebrafish provoked further enhancements in the expression of α6 and α7 subunit, Pitx3, and TH1 mRNAs suggesting that the expression of these molecules in the reward pathway is involved in both processes. Our findings indicate that repeated exposures to low doses of drugs of abuse can increase subject's sensitivity to the rewarding properties of the same or different drugs. This further suggests that casual drug intake increases the probability of becoming addict.


Assuntos
Sensibilização do Sistema Nervoso Central/efeitos dos fármacos , Cocaína/farmacologia , Condicionamento Clássico/efeitos dos fármacos , Nicotina/farmacologia , Receptores Nicotínicos/biossíntese , Peixe-Zebra , Animais , Encéfalo/metabolismo , Proteínas de Homeodomínio/biossíntese , Locomoção/efeitos dos fármacos , Tirosina 3-Mono-Oxigenase/biossíntese , Proteínas de Peixe-Zebra/biossíntese
12.
Birth Defects Res ; 111(12): 775-788, 2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-30648819

RESUMO

BACKGROUND: Recent work suggests that endocannabinoids (eCBs) may signal through the sonic hedgehog signaling pathway. We therefore hypothesized that combined ethanol and eCB exposure during defined stages of zebrafish embryogenesis will produce deficits comparable to human fetal alcohol spectrum disorder (FASD). METHODS: Zebrafish embryos were exposed to ethanol or cannabinoid agonists alone or in combination at defined developmental stages and assessed for changes in brain morphology or expression of marker genes such as pax6a. Juvenile fish were then assessed for risk-taking/anxiety-like behavior using the novel tank dive test. RESULTS: Either chronic or acute exposure to high doses of the CB1R agonist ACEA resulted in FASD phenotypes. However, acute subthreshold doses of CB1R agonist alone, or combined with 0.5% ethanol, did not induce morphological phenotypes, but did induce dysmorphogenesis when combined with acute 1% ethanol. Phenotypes were rescued using the CB1R antagonist SR141716A. In addition, JZL195, a dual inhibitor of FAAH and MAGL, two degradative enzymes for eCBs, induced FASD phenotypes in the presence of subthreshold ethanol, confirming the activation of common signaling pathways by ethanol and eCBs. We next analyzed the effects of ethanol and CB1R agonist on juvenile zebrafish behavior and show that ACEA or ethanol alone did not alter behavior, but combined ACEA and ethanol increased risk-taking behavior. CONCLUSIONS: These studies demonstrate that pathological and behavioral phenotypes associated with FASD are induced by exposure to CB1R agonists and suggest that combined exposure to lower levels of alcohol and marijuana may be capable of inducing FASD-like morphological and behavioral impairments.


Assuntos
Canabinoides/efeitos adversos , Embrião não Mamífero/embriologia , Etanol/efeitos adversos , Transtornos do Espectro Alcoólico Fetal/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Proteínas de Peixe-Zebra/biossíntese , Peixe-Zebra/embriologia , Animais , Canabinoides/farmacologia , Modelos Animais de Doenças , Embrião não Mamífero/patologia , Etanol/farmacologia , Transtornos do Espectro Alcoólico Fetal/patologia , Transtornos do Espectro Alcoólico Fetal/fisiopatologia
13.
Appl Immunohistochem Mol Morphol ; 27(3): 174-179, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-28800015

RESUMO

Although molecular testing can definitively distinguish Ewing sarcoma (EWS) from synovial sarcoma (SS) it is frequently desirable to provide a confident preliminary diagnosis before such analysis can be completed. Recently, the nuclear markers NKX2.2 and TLE1 have been shown to have good sensitivity but imperfect specificity, respectively, for EWS and SS. However, the performance of these markers has not been extensively evaluated within this specific differential diagnosis. This study performed NKX2.2, TLE1, and CD99 immunohistochemistry in a group of EWS and SSs confirmed by reverse transcription-polymerase chain reaction to evaluate the utility of these novel markers in this context. NKX2.2 staining was overall 75% sensitive and 91.7% specific for EWS and was never seen in SS. Although the specificity of TLE1 staining was impacted by antibody used, it was at best only 75% specific for SS. However, a lack of reactivity had a 100% negative predictive value against a SS diagnosis. Overall, immunohistochemistry for NKX2.2 and TLE1 can provide a useful first step in helping to distinguish EWS and SS.


Assuntos
Biomarcadores Tumorais/biossíntese , Neoplasias Ósseas , Proteínas Correpressoras/biossíntese , Proteínas de Homeodomínio/biossíntese , Proteínas de Neoplasias/biossíntese , Sarcoma de Ewing , Sarcoma Sinovial , Proteínas de Peixe-Zebra/biossíntese , Adolescente , Adulto , Idoso , Neoplasias Ósseas/diagnóstico , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Criança , Pré-Escolar , Diagnóstico Diferencial , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Sarcoma de Ewing/diagnóstico , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patologia , Sarcoma Sinovial/diagnóstico , Sarcoma Sinovial/metabolismo , Sarcoma Sinovial/patologia
14.
Cancer Cytopathol ; 126(11): 942-949, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30376220

RESUMO

BACKGROUND: Ewing sarcoma (ES) is a round cell sarcoma that can be challenging to diagnose on cytologic material given its significant overlap with numerous mesenchymal, epithelial, and lymphoid cytomorphologic mimics. The objective of this study was to assess the utility of a novel marker, NKX2.2, in the diagnosis of ES in cytologic material and its ability to distinguish ES from its mimics. METHODS: NKX2.2 immunohistochemistry was performed on cell blocks from 107 fine-needle aspirations, and nuclear expression was scored semiquantitatively for extent and intensity. The study cohort included ES (n = 10), well differentiated neuroendocrine tumor (n = 20), melanoma (n = 11), Merkel cell carcinoma (n = 10), small cell carcinoma (n = 10), alveolar rhabdomyosarcoma (n = 2), spindle cell/sclerosing rhabdomyosarcoma (n = 2), synovial sarcoma (n = 12), solitary fibrous tumor (n = 2), chronic lymphocytic leukemia (n = 10), lymphoblastic lymphoma (n = 11), adenoid cystic carcinoma (n = 6), and CIC-rearranged sarcoma (n = 1). RESULTS: NKX2.2 had high sensitivity (100%) and moderate specificity (85%) for the diagnosis of ES in cytologic material. NKX2.2 expression also was present in a subset of mesenchymal and epithelial mimics, and staining was most commonly observed in small cell carcinoma (80%) and well differentiated neuroendocrine tumor (45%). Among mesenchymal mimics, 42% exhibited NKX2.2 expression. NKX2.2 staining was absent in melanoma, adenoid cystic carcinoma, and lymphoproliferative neoplasms. CONCLUSIONS: NKX2.2 is a highly sensitive but only moderately specific marker for ES. Neuroendocrine neoplasms exhibit variable NKX2.2 expression and remain a significant potential diagnostic pitfall. Thus, NKX2.2 expression should be interpreted in the context of an appropriate immunohistochemical panel (and often with confirmatory molecular testing) for the accurate diagnosis of ES.


Assuntos
Biomarcadores Tumorais/biossíntese , Biópsia por Agulha Fina/métodos , Proteínas de Homeodomínio/biossíntese , Imuno-Histoquímica/métodos , Sarcoma de Ewing/patologia , Proteínas de Peixe-Zebra/biossíntese , Carcinoma de Célula de Merkel/metabolismo , Carcinoma de Célula de Merkel/patologia , Carcinoma de Células Pequenas/metabolismo , Carcinoma de Células Pequenas/patologia , Diagnóstico Diferencial , Humanos , Melanoma/metabolismo , Melanoma/patologia , Tumores Neuroendócrinos/metabolismo , Tumores Neuroendócrinos/patologia , Reprodutibilidade dos Testes , Estudos Retrospectivos , Sarcoma de Ewing/metabolismo , Sarcoma de Células Pequenas/metabolismo , Sarcoma de Células Pequenas/patologia , Sensibilidade e Especificidade
15.
Development ; 145(17)2018 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-30082270

RESUMO

Functional analyses of genes responsible for neurodegenerative disorders have unveiled crucial links between neurodegenerative processes and key developmental signalling pathways. Mutations in SPG4-encoding spastin cause hereditary spastic paraplegia (HSP). Spastin is involved in diverse cellular processes that couple microtubule severing to membrane remodelling. Two main spastin isoforms are synthesised from alternative translational start sites (M1 and M87). However, their specific roles in neuronal development and homeostasis remain largely unknown. To selectively unravel their neuronal function, we blocked spastin synthesis from each initiation codon during zebrafish development and performed rescue analyses. The knockdown of each isoform led to different motor neuron and locomotion defects, which were not rescued by the selective expression of the other isoform. Notably, both morphant neuronal phenotypes were observed in a CRISPR/Cas9 spastin mutant. We next showed that M1 spastin, together with HSP proteins atlastin 1 and NIPA1, drives motor axon targeting by repressing BMP signalling, whereas M87 spastin acts downstream of neuropilin 1 to control motor neuron migration. Our data therefore suggest that defective BMP and neuropilin 1 signalling may contribute to the motor phenotype in a vertebrate model of spastin depletion.


Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Neurônios Motores/citologia , Neuropilina-1/metabolismo , Espastina/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/embriologia , Animais , Axônios/metabolismo , Células COS , Sistemas CRISPR-Cas/genética , Linhagem Celular , Movimento Celular/genética , Chlorocebus aethiops , Proteínas de Ligação ao GTP/metabolismo , Técnicas de Inativação de Genes , Humanos , Proteínas de Membrana/metabolismo , Isoformas de Proteínas/genética , Paraplegia Espástica Hereditária/genética , Espastina/biossíntese , Proteínas de Peixe-Zebra/biossíntese
16.
PLoS One ; 13(8): e0201278, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30070994

RESUMO

Disproportionate high intake of n-6 polyunsaturated fatty acids (PUFAs) in the diet is considered as a major human health concern. The present study examines changes in the hepatic gene expression pattern of adult male zebrafish progeny associated with high levels of the n-6 PUFA arachidonic acid (ARA) in the parental diet. The parental generation (F0) was fed a diet which was either low (control) or high in ARA (high ARA). Progenies of both groups (F1) were given the control diet. No differences in body weight were found between the diet groups within adult stages of either F0 or F1 generation. Few differentially expressed genes were observed between the two dietary groups in the F0 in contrast to the F1 generation. Several links were found between the previous metabolic analysis of the parental fish and the gene expression analysis in their adult progeny. Main gene expression differences in the progeny were observed related to lipid and retinoid metabolism by PPARα/RXRα playing a central role in mediating changes to lipid and long-chain fatty acid metabolism. The enrichment of genes involved in ß-oxidation observed in the progeny, corresponded to the increase in peroxisomal ß-oxidative degradation of long-chain fatty acids in the parental fish metabolomics data. Similar links between the F0 and F1 generation were identified for the methionine cycle and transsulfuration pathway in the high ARA group. In addition, estrogen signalling was found to be affected by parental high dietary ARA levels, where gene expression was opposite directed in F1 compared to F0. This study shows that the dietary n-3/n-6 PUFA ratio can alter gene expression patterns in the adult progeny. Whether the effect is mediated by permanent epigenetic mechanisms regulating gene expression in developing gametes needs to be further investigated.


Assuntos
Ácido Araquidônico/farmacologia , Gorduras na Dieta/farmacologia , Fígado/metabolismo , Transcriptoma/efeitos dos fármacos , Proteínas de Peixe-Zebra/biossíntese , Peixe-Zebra/metabolismo , Animais
17.
Development ; 145(13)2018 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-29945867

RESUMO

Neuroendocrine cells in the pineal gland release melatonin during the night and, in teleosts, are directly photoreceptive. During development of the pineal complex, a small number of cells migrate leftward away from the pineal anlage to form the parapineal cell cluster, a process that is crucial for asymmetrical development of the bilateral habenular nuclei. Here, we show that, throughout zebrafish embryonic development, the brain-specific homeobox (bsx) gene is expressed in all cell types of the pineal complex. We identified Bmp and Noto/Flh as major regulators of bsx expression in the pineal complex. Upon loss of Bsx through the generation of a targeted mutation, embryos fail to form a parapineal organ and develop right-isomerized habenulae. Crucial enzymes in the melatonin biosynthesis pathway are not expressed, suggesting the absence of melatonin from the pineal gland in bsx mutants. Several genes involved in rod-like or cone-like phototransduction are also abnormally expressed, indicating that Bsx has a pivotal role in the differentiation of multiple cell types in the zebrafish pineal complex.


Assuntos
Diferenciação Celular/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Homeodomínio/biossíntese , Glândula Pineal/embriologia , Proteínas de Peixe-Zebra/biossíntese , Peixe-Zebra/embriologia , Animais , Proteínas de Homeodomínio/genética , Melatonina/biossíntese , Melatonina/genética , Glândula Pineal/citologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
18.
PLoS One ; 13(6): e0199777, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29940038

RESUMO

BACKGROUND: TGF-ß signaling is a cellular pathway that functions in most cells and has been shown to play a role in multiple processes, such as the immune response, cell differentiation and proliferation. Recent evidence suggests a possible interaction between TGF-ß signaling and the molecular circadian oscillator. The current study aims to characterize this interaction in the zebrafish at the molecular and behavioral levels, taking advantage of the early development of a functional circadian clock and the availability of light-entrainable clock-containing cell lines. RESULTS: Smad3a, a TGF-ß signaling-related gene, exhibited a circadian expression pattern throughout the brain of zebrafish larvae. Both pharmacological inhibition and indirect activation of TGF-ß signaling in zebrafish Pac-2 cells caused a concentration dependent disruption of rhythmic promoter activity of the core clock gene Per1b. Inhibition of TGF-ß signaling in intact zebrafish larvae caused a phase delay in the rhythmic expression of Per1b mRNA. TGF-ß inhibition also reversibly disrupted, phase delayed and increased the period of circadian rhythms of locomotor activity in zebrafish larvae. CONCLUSIONS: The current research provides evidence for an interaction between the TGF-ß signaling pathway and the circadian clock system at the molecular and behavioral levels, and points to the importance of TGF-ß signaling for normal circadian clock function. Future examination of this interaction should contribute to a better understanding of its underlying mechanisms and its influence on a variety of cellular processes including the cell cycle, with possible implications for cancer development and progression.


Assuntos
Relógios Circadianos/fisiologia , Regulação da Expressão Gênica/fisiologia , Proteínas Circadianas Period/biossíntese , Transdução de Sinais/fisiologia , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Proteínas de Peixe-Zebra/biossíntese , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Linhagem Celular , Feminino , Masculino , Proteínas Circadianas Period/genética , Proteína Smad3/genética , Fator de Crescimento Transformador beta/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
19.
Development ; 145(11)2018 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-29752384

RESUMO

During zebrafish fin regeneration, blastema cells lining the epidermis differentiate into osteoblasts and joint cells to reconstruct the segmented bony rays. We show that osteoblasts and joint cells originate from a common cell lineage, but are committed to different cell fates. Pre-osteoblasts expressing runx2a/b commit to the osteoblast lineage upon expressing sp7, whereas the strong upregulation of hoxa13a correlates with a commitment to a joint cell type. In the distal regenerate, hoxa13a, evx1 and pthlha are sequentially upregulated at regular intervals to define the newly identified presumptive joint cells. Presumptive joint cells mature into joint-forming cells, a distinct cell cluster that maintains the expression of these factors. Analysis of evx1 null mutants reveals that evx1 is acting upstream of pthlha and downstream of or in parallel with hoxa13a Calcineurin activity, potentially through the inhibition of retinoic acid signaling, regulates evx1, pthlha and hoxa13a expression during joint formation. Furthermore, retinoic acid treatment induces osteoblast differentiation in mature joint cells, leading to ectopic bone deposition in joint regions. Overall, our data reveal a novel regulatory pathway essential for joint formation in the regenerating fin.


Assuntos
Nadadeiras de Animais/crescimento & desenvolvimento , Calcineurina/metabolismo , Articulações/crescimento & desenvolvimento , Regeneração/fisiologia , Tretinoína/farmacologia , Peixe-Zebra/fisiologia , Animais , Diferenciação Celular/fisiologia , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/genética , Osteoblastos/citologia , Proteína Relacionada ao Hormônio Paratireóideo/biossíntese , Proteína Relacionada ao Hormônio Paratireóideo/genética , Fator de Transcrição Sp7/biossíntese , Fator de Transcrição Sp7/genética , Fatores de Transcrição/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/biossíntese , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
20.
Adv Exp Med Biol ; 1074: 387-393, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29721968

RESUMO

Factor XIII-A (FXIII-A), which has become known as cellular transglutaminase, plays important roles in mediating cross-linking reactions in various tissues. FXIII-A acts as one of the regeneration molecules in the fish retina and optic nerve after optic nerve injury and becomes activated at the site of injury within a few hours. Previous research has shown that activated FXIII-A induces neurite outgrowth from injured retinal ganglion cells and supports elongation of the regenerating optic nerve. However, the activation mechanism of FXIII-A remains unknown. Furthermore, the injured tissues do not express thrombin, a known activator of plasma FXIII. Here, we investigated the mRNA expression of FXIII-A based on two different regions, one encoding the activation peptide and the other encoding the enzymatic active site. We found that expression of the region encoding the activation peptide was markedly suppressed compared with the region encoding the active site. An overexpression study with a short-type FXIII-A cDNA lacking the activation peptide revealed induction of long neurite outgrowth in fish retinal explant cultures compared with full-length FXIII-A cDNA. The present findings suggest that alternative splicing may occur in the FXIII-A gene, resulting in deletion of the region encoding the activation peptide and thus allowing direct production of activated FXIII-A protein in the fish retina and optic nerve after optic nerve injury.


Assuntos
Processamento Alternativo , Proteínas do Olho/genética , Fator XIIIa/metabolismo , Traumatismos do Nervo Óptico/genética , RNA Mensageiro/genética , Proteínas de Peixe-Zebra/genética , Animais , Axônios/ultraestrutura , Ativação Enzimática , Proteínas do Olho/biossíntese , Proteínas do Olho/fisiologia , Regulação da Expressão Gênica , Carpa Dourada , Peptídeos e Proteínas de Sinalização Intercelular , Compressão Nervosa , Regeneração Nervosa , Traumatismos do Nervo Óptico/metabolismo , Técnicas de Cultura de Órgãos , Peptídeos/metabolismo , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Peixe-Zebra , Proteínas de Peixe-Zebra/biossíntese , Proteínas de Peixe-Zebra/fisiologia
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