Neurotoxicity of methylmercury in Drosophila embryo development

说明(申请人提供)：甲基汞(MeHg)是一种持久性环境神经毒素，由于其在食用鱼类中积累，对人类健康构成风险。流行病学和实验室研究证实，发育中的神经系统对甲基汞毒性特别敏感。我们的总体目标是阐明甲基汞毒性所针对的神经发育的基本机制。甲基汞选择性地改变神经发生与细胞命运、神经元迁移和/或神经元形态的程度尚不清楚。然而，众所周知，这些神经发育事件在胚胎发育过程中是有时间调节的。我们的假设是，对于四种神经发育事件中的一种，甲基汞将表现出更高的活性：神经母细胞规格、神经元兄弟细胞命运、神经元/神经胶质细胞迁移或神经元形态发生。因此，我们预测胚胎神经系统对甲基汞毒性的敏感性将随着甲基汞暴露的发育时间而变化。我们将用果蝇胚胎模型来检验这一假设。我们的研究之所以可行，是因为我们突破性地创新了胚胎渗透溶剂(EPS)，该溶剂克服了长期存在的技术障碍，即在保持胚胎活力的同时渗透果蝇蛋壳。EPS治疗能够将规定剂量的小分子输送到体外培养的胚胎中。通过将甲基汞应用于果蝇胚胎的离散发育时间点，并监测具有良好特征的神经谱系的表型，我们期望阐明早期(神经发生和细胞命运指定)或晚期(神经元迁移和形态发生)发育事件对甲基汞毒性最敏感。由于EPS是一种新的应用，我们将首先采取措施校准胚胎的渗透性水平，并对处理后的胚胎进行甲基汞剂量测定。然后，我们将把这一方法应用于现有的带有谱系特异性报告基因的转基因果蝇品系，以确定最脆弱的神经发育机制。由于果蝇胚胎神经发育的许多信号通路是高度保守的，我们预计这些结果将推动对高等生物体和人类中的甲基汞靶标进行更集中的分子研究。我们新颖的胚胎透过性创新是在对苍蝇胚胎执行准确剂量方面的使能步骤。因此，我们也认为这项研究对于建立这种实验方法对更广泛的毒理学研究社区的效用至关重要。 公共卫生意义：甲基汞是一种环境神经毒素，会破坏胎儿和幼儿的神经系统发育。这项建议试图确定胚胎发生过程中神经发育最易受甲基汞影响的机制。对这些机制的了解将提高我们确定和建议暴露在甲基汞中风险最高的人的能力。

DESCRIPTION (provided by applicant): Methylmercury (MeHg) is a persistent environmental neurotoxin that poses a health risk to humans due to its accumulation in dietary fish. Epidemiological and laboratory studies have established that the developing nervous system is exceptionally sensitive to MeHg toxicity. Our overall goal is to elucidate the fundamental mechanisms of neural development that are targeted by MeHg toxicity. The extent to which MeHg selectively alters neurogenesis versus cell fates, neuronal migration and/or neuron morphology remains unclear. It is well understood, however, that these neural developmental events are temporally regulated over the course of embryogenesis. Our hypothesis is that MeHg will show a preferentially higher activity toward disruption of one of four neural developmental events: neuroblast specification, neuron sibling cell fates, neuronal/glial migration or neuron morphogenesis. We therefore predict that susceptibility of the embryonic nervous system to MeHg toxicity will vary with the developmental timing of MeHg exposure. We will test this hypothesis with the Drosophila embryo model. Our study is made feasible by our breakthrough innovation of an embryo permeabilization solvent (EPS) that overcomes the longstanding technical barrier of permeating of the fruit fly eggshell while maintaining embryo viability. EPS treatment enables delivery of defined doses of small molecules to embryos cultured in vitro. By applying MeHg to the Drosophila embryo at discrete developmental time points and monitoring phenotypes in well characterized neural lineages we expect to elucidate whether early (neurogenesis and cell fate specification) or late (neuron migration and morphogenesis) development events are the most susceptible to MeHg toxicity. As EPS is a new application we will first take steps to calibrate levels of embryo permeability and perform MeHg dosimetry with treated embryos. We will then apply this methodology to available transgenic fly strains with lineage-specific reporter genes to determine the most vulnerable neural developmental mechanisms. Since many of the signaling pathways underlying neural development in the fly embryo are highly conserved, we expect these results will drive the rationale for more focused molecular studies on MeHg targets in higher organisms and in humans. Our novel embryo permeabilization innovation is an enabling step in executing accurate doses to the fly embryo. Therefore, we also view this study as essential for establishing the utility of this experimental approach for the broader toxicological research community. PUBLIC HEALTH RELEVANCE: Methylmercury is an environmental neurotoxin that disrupts development of the nervous system in the fetus and young children. This proposal seeks to identify the most methylmercury-susceptible mechanisms of neural development during the course of embryogenesis. Knowledge of these mechanisms will advance our ability to determine and advise people who are most at risk when exposed to methylmercury.