Methylmercury-induced hair cell loss requires hydrogen peroxide production and leukocytes in zebrafish embryos.

Hearing impairment and deafness is frequently observed as one of the neurological signs in patients with Minamata disease caused by methylmercury (MeHg) poisoning. Loss of hair cells in humans and animals is a consequence of MeHg poisoning. However, it is still not clear how MeHg causes hearing deficits. We employed the hair cells of the lateral line system of zebrafish embryos as a model to explore this question. We exposed transgenic zebrafish embryos to MeHg (30-360 µg/L) at the different stages, and scored the numbers of hair cells. We find that MeHg-induced reduction of hair cells is in a concentration dependent manner. By employing antisense morpholino against to pu.1, we confirm that loss of hair cells involves the action of leukocytes. Moreover, hair cell loss is attenuated by co-treating MeHg-exposed embryos with pharmacological inhibitors of NADPH oxidases named diphenyleneiodonium (DPI) and VAS2870. In situ gene expression analysis showed that genes encoding the SQSTM1-Keap1-Nrf2 systems involved in combating oxidative stress and immune responses are highly expressed in the lateral line organs of embryos exposed to MeHg. This suggests that induction of hydrogen peroxide (H2O2) is the primary effect of MeHg on the hair cells. Genes induced by MeHg are also involved in regeneration of the hair cells. These features are likely related to the capacity of the zebrafish to regenerate the lost hair cells.

Melanosomes in pigmented epithelia maintain eye lens transparency during zebrafish embryonic development.

Altered levels of trace elements are associated with increased oxidative stress that is eventually responsible for pathologic conditions. Oxidative stress has been proposed to be involved in eye diseases, including cataract formation. We visualized the distribution of metals and other trace elements in the eye of zebrafish embryos by micro X-ray fluorescence (µ-XRF) imaging. Many elements showed highest accumulation in the retinal pigment epithelium (RPE) of the zebrafish embryo. Knockdown of the zebrafish brown locus homologues tyrp1a/b eliminated accumulation of these elements in the RPE, indicating that they are bound by mature melanosomes. Furthermore, albino (slc45a2) mutants, which completely lack melanosomes, developed abnormal lens reflections similar to the congenital cataract caused by mutation of the myosin chaperon Unc45b, and an in situ spin trapping assay revealed increased oxidative stress in the lens of albino mutants. Finally transplanting a wildtype lens into an albino mutant background resulted in cataract formation. These data suggest that melanosomes in pigment epithelial cells protect the lens from oxidative stress during embryonic development, likely by buffering trace elements.

Gene responses in the central nervous system of zebrafish embryos exposed to the neurotoxicant methyl mercury.

Methyl mercury (MeHg) is a neurotoxicant with adverse effects on the development of the nervous system from fish to man. Despite a detailed understanding of the molecular mechanisms by which MeHg affects cellular homeostasis, it is still not clear how MeHg causes developmental neurotoxicity. We performed here a genome-wide transcriptional analysis of MeHg-exposed zebrafish embryos and combined this with a whole-mount in situ expression analysis of 88 MeHg-affected genes. The majority of the analyzed genes showed tissue- and region-restricted responses in various organs and tissues. The genes were linked to gene ontology terms like oxidative stress, transport and cell protection. Areas even within the central nervous system (CNS) are affected differently resulting in distinct cellular stress responses. Our study revealed an unexpected heterogeneity in gene responses to MeHg exposure in different tissues and neuronal subregions, even though the known molecular action of MeHg would predict a similar burden of exposed cells. The overall structure of the developing brain of MeHg-exposed embryos appeared normal, suggesting that the mechanism leading to differentiation of the CNS is not overtly affected by exposure to MeHg. We propose that MeHg disturbs the function of the CNS by disturbing the cellular homeostasis. As these cellular stress responses comprise genes that are also involved in normal neuronal activity and learning, MeHg may affect the developing CNS in a subtle manner that manifests itself in behavioral deficits.

Methyl mercury suppresses the formation of the tail primordium in developing zebrafish embryos.

The objective of this study was to characterize the mechanisms of action of the model environmental toxicant methyl mercury (MeHg) in the zebrafish embryo. Zebrafish embryos were exposed to MeHg, and the effective concentration and window of exposure were determined in wild-type and fluorescent reporter transgenic zebrafish embryos. Genes were systematically assessed for altered expression in response to MeHg by in situ hybridization. MeHg impairs development of the fin fold and the tail fin primordium. Alterations in transgene expression were noted at 6 microg/l MeHg, making this shh:gfp line the most sensitive biosensor of MeHg exposure. The matrix metalloproteases mmp9 and mmp13 and eight other genes are induced in the embryonic tail in response to MeHg. Our data suggest that MeHg impairs tail development at least partially by activation of the tissue remodeling proteases Mmp9 and Mmp13.

Zebrafish embryos as models for embryotoxic and teratological effects of chemicals.

The experimental virtues of the zebrafish embryo such as small size, development outside of the mother, cheap maintenance of the adult made the zebrafish an excellent model for phenotypic genetic and more recently also chemical screens. The availability of a genome sequence and several thousand mutants and transgenic lines together with gene arrays and a broad spectrum of techniques to manipulate gene functions add further to the experimental strength of this model. Pioneering studies suggest that chemicals can have in many cases very similar toxicological and teratological effects in zebrafish embryos and humans. In certain areas such as cardiotoxicity, the zebrafish appears to outplay the traditional rodent models of toxicity testing. Several pilot projects used zebrafish embryos to identify new chemical entities with specific biological functions. In combination with the establishment of transgenic sensor lines and the further development of existing and new automated imaging systems, the zebrafish embryos could therefore be used as cost-effective and ethically acceptable animal models for drug screening as well as toxicity testing.

Cloning and developmental expression of kinesin superfamily7 (kif7) in the brackish medaka (Oryzias melastigma), a close relative of the Japanese medaka (Oryzias latipes).

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.