Zebrafish as a model to study the neuroendocrine system and toxicity of endocrine disruptors

摘要

Zebrafish is a popular vertebrate model system to study development andperform genetic analysis. It offers numerous advantages such as small size, shortgeneration time, high fecundity, rapid and ex utero development of embryos andoptically transparent embryos. Zebrafish is genetically closely related to humans andshare high similarity in developmental processes, physiology and behavior. In addition,recent advances in forward and reverse genetics coupled with the availability of a largenumber of mutants makes zebrafish a useful model for genetic analysis of development.Furthermore, the above mentioned advantages offered by this model organism will be avaluable resource for toxicologists not only to assess toxicity of endocrine disruptingchemicals (EDCs) but also to dissect the mechanisms of toxicity of EDCs.The development and function of the neuroendocrine system in zebrafishis to a great extent similar to other vertebrates. Thus, in an aim to understand thedevelopment of the neuroendocrine stress axis we have characterized the expression ofcorticotropin-releasing hormone (crh) in the embryonic brain of zebrafish. Transcriptsof crh were detected in a broad range of adult tissues and also during embryonic andlarval stages. Whole-mount in situ hybridization analysis revealed distribution of crhtranscripts in various regions of the developing brain including telencephalon, preopticarea, hypothalamus, posterior tuberculum, thalamus, epiphysis, midbrain tegmentum,hindbrain and retina. Expression of crh in the preoptic area and in extra-hypothalamicregions is consistent with its roles as a hypophysiotropin and a neuromodulator.Estrogen receptors are ligand activated transcription factors involved inregulating the neuroendocrine axis of reproduction. We have analyzed the mRNAlevels of the esr genes in the absence and presence of exogenous ligands. The three esrgenes, esr1 (ERalpha), esr2b (ERbeta1) and esr2a (ERbeta2) were expressed in alladult tissues tested in the absence of exogenous ligands. Expressions of esr1 and esr2awere altered in the liver, brain, testis and intestine following ligand treatment (17β-estradiol (E2) or diethylstilbestrol (DES) or 4-nonylphenol, (4-NP)). Duringembryogenesis, only esr1 and esr2b were predominantly expressed and both wereregulated by ligands. Our results demonstrate that the esr genes in zebrafish areregulated in a sex- and tissue specific manner and that 4-NP, a well-known endocrinedisruptor possess both agonist and antagonist properties in adult tissues.We further extended our study to assess the toxic effects of 4-NP ondeveloping embryos and larvae of zebrafish. A sub-lethal dose of 4-NP not onlyperturbed the neuroendocrine axis but also induced distortions/kinks and herniations inthe notochord. The differentiation of the notochordal cells and the formation of theperinotochordal basement membrane were disrupted by 4-NP. Early disturbancesinduced by 4-NP in the notochord resulted in deformaties in the vertebral column at latelarval stages. The notochord phenotype was accompanied by impaired swimmingpattern. Repeated electrical stimulation of the larval muscles of 4-NP treated embryosshowed impairment in the relaxation between stimuli which might be a possible reasonfor the defective swimming observed in 4-NP treated embryos.