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）是一种有充分记录的发育神经毒性物质，但导致甲基汞诱导神经毒性的精确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系统发育受损，并导致各种缺陷，从严重智力迟钝、脑瘫和失明到运动功能、感觉反应以及学习和记忆方面的轻微缺陷。我们的实验室已经证明，成年斑马鱼在发育过程中暴露于与人类暴露水平相当的甲基汞，也会出现同样的神经行为异常。我们实验室的长期目标是通过有效建模， 表征和了解斑马鱼模型系统中发育期汞暴露的影响。适当的神经行为反应需要所有神经系统组成部分的功能整合，而异常强烈表明神经系统发育过程中建立的基本解剖学或细胞生理学的改变。我们已经表明，斑马鱼发育暴露于甲基汞表现出神经行为异常，包括多动症以及在视觉，听觉，学习和记忆的缺陷。该项目的总体目标是确定和表征关键的，敏感的神经元细胞populatons改变发育中的甲基汞暴露在整个动物的背景下。我们的假设是，发育中暴露于甲基汞，在浓度远低于导致明显的毒性，结果在神经解剖结构和基因表达谱的特定和关键的细胞群在发育中的神经系统的变化。与拟议的研究，我们将扩大我们的斑马鱼模型的甲基汞诱导的神经发育毒性，通过使用转基因斑马鱼表达细胞特异性荧光报告蛋白，以确定和表征独特的人口改变发育甲基汞暴露。该项目的具体目的是在脑内鉴定特定的甲基汞敏感神经元群体。 通过表征神经解剖学异常和改变基因表达谱引起的发育甲基汞暴露在斑马鱼模型系统的神经系统。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的特异性荧光标记细胞群进行详细分析，以及荧光激活细胞分选（FACS），然后对荧光素阳性细胞进行RNA-seq转录组分析来实现。鉴于约15%的美国育龄妇女的血汞浓度高于预期导致敏感儿童发育缺陷的水平，因此在基本生物学水平以及整个动物的背景下了解甲基汞诱导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.