RESUMEN

The negative effects of overexposure to ultraviolet (UV) radiation in humans, including sunburn and light-induced cellular injury, are of increasing public concern. 4-Methylbenzylidene camphor (4-MBC), an organic chemical UV filter, is an active ingredient in sunscreen products. To date, little information is available about its neurotoxicity during early vertebrate development. Zebrafish embryos were exposed to various concentrations of 4-MBC in embryo medium for 3 days. In this study, a high concentration of 4-MBC, which is not being expected at the current environmental concentrations in the environment, was used for the purpose of phenotypic screening. Embryos exposed to 15 µM of 4-MBC displayed abnormal axial curvature and exhibited impaired motility. Exposure effects were found to be greatest during the segmentation period, when somite formation and innervation occur. Immunostaining of the muscle and axon markers F59, znp1, and zn5 revealed that 4-MBC exposure leads to a disorganized pattern of slow muscle fibers and axon pathfinding errors during the innervation of both primary and secondary motor neurons. Our results also showed reduction in AChE activity upon 4-MBC exposure both in vivo in the embryos (15 µM) and in vitro in mammalian Neuro-2A cells (0.1 µM), providing a possible mechanism for 4-MBC-induced muscular and neuronal defects. Taken together, our results have shown that 4-MBC is a teratogen and influences muscular and neuronal development, which may result in developmental defects.

Asunto(s)

Alcanfor/análogos & derivados , Embrión no Mamífero/efectos de los fármacos , Desarrollo de Músculos/efectos de los fármacos , Sistema Nervioso/efectos de los fármacos , Protectores Solares/toxicidad , Pez Cebra/embriología , Animales , Alcanfor/toxicidad , Embrión no Mamífero/fisiología , Humanos , Sistema Nervioso/crecimiento & desarrollo , Rayos Ultravioleta

RESUMEN

Hypoxia can impair reproduction of fishes through the disruption of sex steroids. Here, using zebrafish (Danio rerio) embryos, we investigated (i) whether hypoxia can directly affect steroidogenesis independent of pituitary regulation via modulation of steroidogenic gene expression, and (ii) the role of leptin in hypoxia-induced disruption of steroidogenesis. Exposure of fertilized zebrafish embryos to hypoxia (1.0 mg O(2) L(-1)) from 0-72 h postfertilization (hpf), a developmental window when steroidogenesis is unregulated by pituitary influence, resulted in the up-regulation of cyp11a, cyp17, and 3ß-hsd and the down-regulation of cyp19a. Similar gene expression patterns were observed for embryos exposed to 10 mM cobalt chloride (CoCl(2), a chemical inducer of hypoxia-inducible factor 1, HIF-1), suggesting a regulatory role of HIF-1 in steroidogenesis. Testosterone (T) and estradiol (E2) concentrations in hypoxic embryos were greater and lesser, respectively, relative to the normoxic control, thus leading to an increased T/E2 ratio. Expression of the leptin-a gene (zlep-a) was up-regulated upon both hypoxia and CoCl(2) treatments. Functional assays suggested that under hypoxia, elevated zlep-a expression might activate cyp11a and 3ß-hsd and inhibit cyp19a. Overall, this study indicates that hypoxia, possibly via HIF-1-induced leptin expression, modulates sex steroid synthesis by acting directly on steroidogenic gene expression.

Asunto(s)

Regulación del Desarrollo de la Expresión Génica , Leptina/fisiología , Pez Cebra/embriología , Animales , Secuencia de Bases , Cartilla de ADN , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Pez Cebra/genética

RESUMEN

In animal retina, hedgehog expression drives waves of neurogenesis, but genetic programs that control its expression during retinal neurogenesis are poorly elucidated. We have previously reported that irx1a is required for propagation of the sonic hedgehog (shh) expression waves in developing zebrafish retina. Here, we found that irx2a is expressed in the developing retina and that knockdown of irx2a results in a retinal phenotype strikingly similar to that of irx1a morphants. The expression of irx2a in retina ganglion cells was shown to be irx1a- and ath5-dependent suggesting that irx1a and ath5 are transcriptional regulators of irx2a. Furthermore, irx2a expression could rescue impaired propagation of shh waves in irx1a morphants. Together, these observations suggest that Irx2 functions downstream of irx1a to control shh expression in the retina. We proposed a novel transcriptional cascade of ath5-irx1a-irx2a in the regulation of hedgehog waves during vertebrate retinal development.

Asunto(s)

Proteínas de Homeodominio/metabolismo , Retina/embriología , Células Ganglionares de la Retina/citología , Factores de Transcripción/metabolismo , Proteínas de Pez Cebra/metabolismo , Animales , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Proteínas de Homeodominio/genética , Factores de Transcripción/genética , Pez Cebra , Proteínas de Pez Cebra/genética

RESUMEN

Hypoxia is the most widespread deleterious consequence of eutrophication and has become a major cause of fishery decline. One feature of chronic exposure to hypoxia in fish is inhibition of feeding. In this study, we investigated if the gene that encodes the appetite-suppressing hormone leptin is regulated by hypoxia in zebrafish (Danio rerio). Exposure of adult zebrafish to hypoxic conditions (1+/-0.2 mg O(2) L(-1)) for 4 and 10 days significantly increased leptin-a (zlep-a) mRNA levels in the liver. To evaluate the role of hypoxia-inducible factor 1 (HIF-1) in regulating zlep-a expression, zebrafish embryos were exposed to cobalt chloride (CoCl(2), a HIF-1 inducer) and overexpressed with HIF-1alpha mRNA. Both CoCl(2) treatment and HIF-1alpha overexpression markedly increased zlep-a expression in developing embryos, indicating the possible involvement of HIF-1 in zlep-a regulation. In vivo promoter analysis indicated that zlep-a promoter activity is found in the muscle fibers of zebrafish embryos and enhanced by CoCl(2). This is the first report to show that leptin gene expression in fish is regulated by hypoxia possibly via the involvement of HIF-1.

Asunto(s)

Hipoxia/metabolismo , Leptina/metabolismo , Hígado/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Animales , Regulación de la Expresión Génica/genética , Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Leptina/genética , Regiones Promotoras Genéticas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Pez Cebra/genética , Proteínas de Pez Cebra/genética

RESUMEN

Fish exposed to hypoxia develop decreased heart rate, or bradycardia, the physiological significance of which remains unknown. The general muscarinic receptor antagonist atropine abolishes the development of this hypoxic bradycardia, suggesting the involvement of muscarinic receptors. In this study, we tested the hypothesis that the hypoxic bradycardia is mediated specifically by stimulation of the M(2) muscarinic receptor, the most abundant subtype in the vertebrate heart. Zebrafish (Danio rerio) were reared at two levels of hypoxia (30 and 40 Torr PO(2)) from the point of fertilization. In hypoxic fish, the heart rate was significantly lower than in normoxic controls from 2 to 10 days postfertilization (dpf). At the more severe level of hypoxia (30 Torr PO(2)), there were significant increases in the relative mRNA expression of M(2) and the cardiac type beta-adrenergic receptors (beta1AR, beta2aAR, and beta2bAR) at 4 dpf. The hypoxic bradycardia was abolished (at 40 Torr PO(2)) or significantly attenuated (at 30 Torr PO(2)) in larvae experiencing M(2) receptor knockdown (using morpholino antisense oligonucleotides). Sham-injected larvae exhibited typical hypoxic bradycardia in both hypoxic regimens. The expression of beta1AR, beta2aAR, beta2bAR, and M(2) mRNA was altered at various stages between 1 and 4 dpf in larvae experiencing M(2) receptor knockdown. Interestingly, M(2) receptor knockdown revealed a cardioinhibitory role for the beta(2)-adrenergic receptor. This is the first study to demonstrate a specific role of the M(2) muscarinic receptor in the initiation of hypoxic bradycardia in fish.

Asunto(s)

Bradicardia/etiología , Corazón/fisiopatología , Hipoxia/complicaciones , Receptor Muscarínico M2/fisiología , Receptores Adrenérgicos beta/metabolismo , Pez Cebra/metabolismo , Antagonistas de Receptores Adrenérgicos beta 1 , Antagonistas de Receptores Adrenérgicos beta 2 , Antagonistas Adrenérgicos beta/farmacología , Estructuras Animales/metabolismo , Animales , Bradicardia/inducido químicamente , Bradicardia/metabolismo , Bradicardia/fisiopatología , Encéfalo/metabolismo , Carbacol/farmacología , Embrión no Mamífero/metabolismo , Expresión Génica/genética , Corazón/efectos de los fármacos , Frecuencia Cardíaca/fisiología , Hipoxia/metabolismo , Hipoxia/fisiopatología , Larva/efectos de los fármacos , Larva/metabolismo , Miocardio/metabolismo , Norepinefrina/farmacología , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Antisentido/genética , Receptores Adrenérgicos beta/genética , Receptores Adrenérgicos beta 1/genética , Receptores Adrenérgicos beta 2/genética

RESUMEN

kif7 is a member of the kinesin superfamily members which are molecular motor proteins that move along microtubules in a highly regulated manner through ATP hydrolysis. In this paper, we report on the cloning of the Oryziasmelastigmakif7 (omkif7) using primers designed according to the Japanese medaka (Oryziaslatipes) database. The cloned omkif7 has an open reading frame of 3762bp and is deduced to encode a polypeptide of 1254 amino acids that possesses the putative ATP-binding and microtubule-binding motifs in its motor domain at the N-terminal region. We characterized the cloned omkif7 by comparison with the zebrafish kif7. Both omkif7 and zebrafish kif7 are shown to be expressed in all embryonic stages and adult tissues examined with higher expression level in the testis and ovary. Whole-mount in situ hybridization revealed that the expression of omkif7 is ubiquitous during the early stages of embryonic development, but became more restrictive and localized to the brain, fin bud and eye at later development. This study suggested that the brackish O.melastigma can serve as a good seawater model organism for developmental studies by utilizing the resources developed from its close relative of the Japanese medaka.

Asunto(s)

Regulación del Desarrollo de la Expresión Génica , Cinesinas/genética , Oryzias/genética , Secuencia de Aminoácidos , Animales , Clonación Molecular , Humanos , Cinesinas/química , Ratones , Datos de Secuencia Molecular , Oryzias/clasificación , Oryzias/embriología , Alineación de Secuencia , Factores de Tiempo

RESUMEN

Chinese materia medica (CMM) is becoming increasingly important in modern health care, with the potential for new or improved clinical protocols and reduction in treatment costs. Conventional approaches to drug discovery are based on knowledge of biological systems and screen phenotypes in the context of a whole organism. It will be valuable to identify the CMM that would induce certain biological responses (such as angiogenesis). The authors have developed a database that they plan to commercialize that contains traditional knowledge of Chinese medicine and pharmacology along with their own experimental data from controlled scientific observations by using the zebrafish as a model of CMM-induced pathology. The database is visualized and functions via the World Wide Web by subscription or license. The authors have also written software for personal digital assistant (PDA) devices that supports multiple users performing screening experiments worldwide. This provides a platform for the study of CMM, and data mining of this resource will help evaluate CMM in the context of experimental observations of biological aberrations.

Asunto(s)

Bases de Datos Factuales , Medicamentos Herbarios Chinos/farmacología , Materia Medica , Programas Informáticos , Animales , Internet , Pez Cebra

RESUMEN

Teleost choriogenins, precursors of the inner layer subunits of the egg envelope, are regarded as sensitive biomarkers for estrogenic pollutants. In this study, two full-length cDNAs, omChgH and omChgL, which encode the choriogenin H and L forms, respectively, were isolated from a brackish medaka, Oryzias melastigma. 17beta-Estradiol (E2; 10 microg/L)-dependent expression of omChgH and omChgL was observed starting at embryonic stage 34 and restricted exclusively to the liver. In hatchlings, E2 induction of omChgH was stronger than that of omChgL. Static exposure of adult fish to E2 (0, 1, 10, 100, and 500 ng/L), 17alpha-ethinylestradiol (EE2; 0, 1, 10, 100 and 500 ng/L), 4-nonylphenol (NP; 0, 1, 10, 100, and 200 microg/L), and bisphenol A (BPA; 0, 1, 10, 100, and 200 microg/L) in artificial seawater for 7 days resulted in dose-dependent induction of both genes in the liver. In the male livers, the sensitivity of omChgH to these estrogenic compounds was higher than that of omChgL; the lowest-observed-effect concentrations (LOECs) of E2, EE2, NP, and BPA on omChgH were 10 ng/L, 10 ng/L, 100 microg/L and 100 microg/L, respectively, and on omChgL were 100 ng/L, 100 ng/L, 100 microg/L, and 200 microg/L, respectively. All these suggest that omChgH can be used as a highly sensitive biomarker for monitoring estrogenic chemicals in the marine environment.

Asunto(s)

Proteínas del Huevo/metabolismo , Estrógenos/farmacología , Proteínas de Peces/metabolismo , Oryzias/metabolismo , Precursores de Proteínas/metabolismo , ARN Mensajero/metabolismo , Contaminantes Químicos del Agua/farmacología , Secuencia de Aminoácidos , Animales , Bioensayo , Biomarcadores , Clonación Molecular , ADN Complementario , Proteínas del Huevo/química , Proteínas del Huevo/genética , Femenino , Proteínas de Peces/química , Proteínas de Peces/genética , Regulación de la Expresión Génica/efectos de los fármacos , Masculino , Datos de Secuencia Molecular , Oryzias/genética , Precursores de Proteínas/química , Precursores de Proteínas/genética , ARN Mensajero/genética , Contaminación del Agua