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

Abstract: Zebrafish is a popular vertebrate model system to study development and perform genetic analysis. It offers numerous advantages such as small size, short generation time, high fecundity, rapid and ex utero development of embryos and optically transparent embryos. Zebrafish is genetically closely related to humans and share high similarity in developmental processes, physiology and behavior. In addition, recent advances in forward and reverse genetics coupled with the availability of a large number of mutants makes zebrafish a useful model for genetic analysis of development. Furthermore, the above mentioned advantages offered by this model organism will be a valuable resource for toxicologists not only to assess toxicity of endocrine disrupting chemicals (EDCs) but also to dissect the mechanisms of toxicity of EDCs. The development and function of the neuroendocrine system in zebrafish is to a great extent similar to other vertebrates. Thus, in an aim to understand the development of the neuroendocrine stress axis we have characterized the expression of corticotropin-releasing hormone (crh) in the embryonic brain of zebrafish. Transcripts of crh were detected in a broad range of adult tissues and also during embryonic and larval stages. Whole-mount in situ hybridization analysis revealed distribution of crh transcripts in various regions of the developing brain including telencephalon, preoptic area, hypothalamus, posterior tuberculum, thalamus, epiphysis, midbrain tegmentum, hindbrain and retina. Expression of crh in the preoptic area and in extra-hypothalamic regions is consistent with its roles as a hypophysiotropin and a neuromodulator. Estrogen receptors are ligand activated transcription factors involved in regulating the neuroendocrine axis of reproduction. We have analyzed the mRNA levels of the esr genes in the absence and presence of exogenous ligands. The three esr genes, esr1 (ERalpha), esr2b (ERbeta1) and esr2a (ERbeta2) were expressed in all adult tissues tested in the absence of exogenous ligands. Expressions of esr1 and esr2a were altered in the liver, brain, testis and intestine following ligand treatment (17β- estradiol (E2) or diethylstilbestrol (DES) or 4-nonylphenol, (4-NP)). During embryogenesis, only esr1 and esr2b were predominantly expressed and both were regulated by ligands. Our results demonstrate that the esr genes in zebrafish are regulated in a sex- and tissue specific manner and that 4-NP, a well-known endocrine disruptor possess both agonist and antagonist properties in adult tissues. We further extended our study to assess the toxic effects of 4-NP on developing embryos and larvae of zebrafish. A sub-lethal dose of 4-NP not only perturbed the neuroendocrine axis but also induced distortions/kinks and herniations in the notochord. The differentiation of the notochordal cells and the formation of the perinotochordal basement membrane were disrupted by 4-NP. Early disturbances induced by 4-NP in the notochord resulted in deformaties in the vertebral column at late larval stages. The notochord phenotype was accompanied by impaired swimming pattern. Repeated electrical stimulation of the larval muscles of 4-NP treated embryos showed impairment in the relaxation between stimuli which might be a possible reason for the defective swimming observed in 4-NP treated embryos.