Identifying the Mechanisms of MeHg-Induced Neurodevelopmental Toxicity using Transgenic Zebrafish

描述(由申请人提供)：尽管甲基汞(MeHg)是一种有充分证据的发育神经毒物，但导致MeHg诱导的神经毒性的确切细胞和分子机制仍然难以捉摸。产前接触甲基汞可导致神经发育受损，并导致各种缺陷，从严重的智力低下、脑瘫和失明到运动功能、感觉反应和学习记忆的轻微缺陷。我们的实验室已经在成年斑马鱼身上证明了同样的神经行为异常，这些斑马鱼在发育过程中暴露在与人类相当的水平的甲基汞中。我们实验室的长期目标是通过有效的建模来确定针对高危人群的潜在干预策略， 在斑马鱼模型系统中表征和理解发育中甲基汞暴露的影响。适当的神经行为反应需要所有神经系统组件的功能整合，而异常强烈地表明在神经系统发育过程中建立的基本解剖学或细胞生理学发生了变化。我们已经证明，在发育过程中暴露于甲基汞的斑马鱼表现出神经行为异常，包括多动以及视觉、听力、学习和记忆方面的缺陷。该项目的总体目标是在整个动物的背景下，识别和表征发育中甲基汞暴露所改变的关键、敏感的神经细胞群体。我们的假设是，发育过程中暴露于甲基汞的浓度远低于导致明显毒性的浓度，会导致发育中神经系统内特定和关键细胞群的神经解剖结构和基因表达谱发生变化。在这项拟议的研究中，我们将通过使用表达细胞特异性荧光报告蛋白的转基因斑马鱼来扩展我们的斑马鱼甲基汞诱导神经发育毒性的模型，以识别和表征发育中甲基汞暴露改变的独特种群。该项目的具体目标是确定特定的甲基汞敏感的神经元群体在 在斑马鱼模型系统中，通过表征发育中甲基汞暴露引起的神经解剖异常和基因表达谱变化来发育神经系统。这一目标将通过使用高分辨率显微镜对发育中的神经系统内特定的荧光标记细胞群进行详细分析，以及随后对报告阳性细胞进行荧光激活细胞分类(FACS)和RNA-seq转录组分析来实现。鉴于美国育龄妇女血汞浓度超过预期会导致敏感儿童发育缺陷的水平，从基础生物学水平以及整个动物的背景了解甲基汞所致神经毒性的机制至关重要。

DESCRIPTION (provided by applicant): Although Methylmercury (MeHg) is a well-documented developmental neurotoxicant, the precise cellular and molecular mechanisms responsible for MeHg-induced neurotoxicity remain elusive. Prenatal MeHg exposure can lead to impaired neurological development and result in a variety of defects ranging from severe mental retardation, cerebral palsy and blindness to subtle deficiencies in motor function, sensory responses, and learning and memory. Our lab has demonstrated this same spectrum of neurobehavioral abnormalities in adult zebrafish that were exposed during development to MeHg at levels comparable to human exposures. The long-term goal of our laboratory is to identify potential intervention strategies for high-risk human populations by effectively modeling, characterizing and understanding the effects of developmental MeHg-exposure in a zebrafish model system. Appropriate neurobehavioral responses require the functional integration of all nervous system components, while abnormalities strongly suggest alterations in basic anatomy or cellular physiology established during nervous system development. We have shown that zebrafish developmentally exposed to MeHg exhibit neurobehavioral abnormalities including hyperactivity as well as deficits in vision, hearing, and learning and memory. The overall objective of this project is to identify and characterize critical, sensitive neuronal cell populatons altered by developmental MeHg exposure in the context of the whole animal. Our hypothesis is that developmental exposure to MeHg, at concentrations well below that which causes overt toxicity, results in changes to the neuroanatomical structure and gene expression profile of specific and critical cell populations within the developing nervous system. With the proposed study, we will expand upon our zebrafish model of MeHg-induced neurodevelopmental toxicity by using transgenic zebrafish expressing cell-specific fluorescent reporter proteins to identify and characterize the unique populations altered by developmental MeHg exposure. The Specific Aim of this project is to identify specific MeHg-sensitive neuronal populations within the developing nervous system through characterization of neuroanatomical abnormalities and altered gene expression profiles induced by developmental MeHg exposure in the zebrafish model system. This Aim will be achieved through a detailed analysis of specific, fluorescently labeled, cell populations within the developing nervous system using high resolution microscopy, and fluorescence-activated cell sorting (FACS) followed by RNA-seq transcriptome analysis of reporter-positive cells. Understanding the mechanism of MeHg-induced neurotoxicity at a basic biological level as well as in the context of the whole animal is critical in light of te ~15% of American women of childbearing age that have blood mercury concentrations above the level expected to cause developmental deficits in sensitive children.