Ahr2 activation and sox9b function in forebrain and cerebral vascular development

描述（由申请人提供）：2,3,7,8 -四氯二苯并-对二恶英（TCDD）是一种持久性亲脂性环境污染物，通过与芳烃受体（AhR）结合而产生毒性。本研究的总体目标是了解暴露于TCDD和其他AhR激动剂如何破坏前脑和脑血管系统的发育，以及最终暴露于AhR激动剂如何导致神经发育障碍和认知障碍。我的初步数据表明，早期胚胎暴露于TCDD严重破坏了斑马鱼前脑和相关脑血管系统的发育。在斑马鱼中可用的遗传和分子工具使其成为研究神经血管发育和毒性的绝佳模型。然而，相对较少的研究使用斑马鱼模型来研究tcdd诱导的Ahr2激活如何影响大脑和脑血管发育。在Aim 1中，使用共聚焦显微镜，荧光免疫组织化学（F-IHC）和Olympus软件，候选人将创建tcdd暴露和对照大脑的3D重建，以详细分析和量化前脑表型。使用AngioTool软件对tcdd引起的脑血管变化进行量化。此外，候选人将使用令人兴奋的新成像技术来检查体内脑血管发育，这将为tcdd诱导的表型如何出现提供独特的见解。在Aim 2中，候选人将以特定细胞类型的方式操纵Ahr2活性，以确定血管内皮细胞或分化神经元中Ahr2的激活是否足以产生所观察到的前脑和脑血管表型。转录因子sox9b的下调已被认为是介导TCDD诱导的心脏、颌和鳍毒性的常见机制。初步数据表明，TCDD暴露后，前脑中sox9b的表达下调，sox9b的缺失导致前脑和脑血管表型与TCDD诱导的前脑和脑血管表型相似。在Aim 3中，候选人将确定sox9b是否也是发育中的大脑中的TCDD靶点，并阐明sox9b在前脑和脑血管系统发育中的功能。重要的是，拟议的研究将帮助我们了解AhR激活如何破坏神经血管发育以及神经血管表型在发育过程中何时出现。这一信息对于了解人类AhR激动剂暴露的潜在风险至关重要。大脑是一个代谢强烈的器官，其正常功能高度依赖于适当和持续的脑血管。因此，这项工作不仅与胚胎发育有关，而且与以后的生命阶段有关。作为K奖获得者，候选人将遵循一个培训计划，该计划概述了在该奖项的过程中要达到的非常具体的研究和职业发展里程碑。为了增强我在神经科学、发育遗传学和心血管毒理学方面的研究背景，我的研究目标是结合神经血管生物学、分子生物学和神经毒理学的教学课程和实践培训。这份培训申请中列出的活动将为我提供成功的研究生涯所需的领域的培训，包括拨款、项目设计和实验室管理。一个由杰出的科学家团队组成的委员会已经成立，致力于候选人和她的成功，并愿意在促进我作为一名科学家的成长中发挥积极的作用

DESCRIPTION (provided by applicant): 2, 3, 7, 8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent, lipophilic environmental contaminant that exerts toxicity through binding the aryl hydrocarbon receptor (AhR). The overall goal of this research is to understand how exposure to TCDD and other AhR agonists disrupts development of the forebrain and cerebral vasculature and, ultimately, how AhR agonist exposure may contribute to neurodevelopmental disorders and cognitive impairment. My preliminary data suggest that early embryonic exposure to TCDD severely disrupts the development of the forebrain and associated cerebral vasculature in zebrafish. The genetic and molecular tools available in zebrafish make it an excellent model for studying neurovascular development and toxicity. However, relatively few studies have used the zebrafish model to examine how TCDD-induced activation of Ahr2 impacts brain and cerebral vascular development. In Aim 1, using confocal microscopy, fluorescent- immunohistochemistry (F-IHC) and Olympus software, the candidate will create 3D reconstructions of TCDD-exposed and control brains for a detailed analysis and quantification of forebrain phenotypes. TCDD-induced changes in the cerebral vasculature will be quantified using AngioTool software. In addition, the candidate will use exciting new imaging technology to examine cerebral vascular development in vivo, which will provide unique insight into how TCDD-induced phenotypes emerge. In Aim 2, the candidate will manipulate Ahr2 activity in a cell-type specific manner to determine if activation of Ahr2 in either vascular endothelial cells or differentiated neurons is sufficient to produce the observed forebrain and cerebral vascular phenotypes. Downregulation of the transcription factor, sox9b, has emerged as a common mechanism mediating TCDD-induced toxicity in the heart, jaw and fin. Preliminary data indicates sox9b expression in the forebrain is downregulated following TCDD exposure and that loss of sox9b results in forebrain and cerebral vascular phenotypes that resemble TCDD-induced forebrain and cerebral vascular phenotypes. In Aim 3, the candidate will determine if sox9b is also a TCDD target in the developing brain and elucidate sox9b function(s) in the development of the forebrain and cerebral vasculature. Importantly, the proposed research will help us understand how AhR activation disrupts neurovascular development and when neurovascular phenotypes emerge during development. This information is essential for understanding potential risks associated with human AhR agonist exposure. The brain is a metabolically intense organ that is highly dependent on proper and continuous cerebral vascularization for normal function. Consequently, this work is relevant not only to embryonic development, but also later life stages. As a K award recipient, the candidate will follow a training plan that outlines very specific research and career development milestones to be reached over the course of the award. To augment my research background in Neuroscience, Developmental Genetics, and Cardiovascular Toxicology, my research aims are coupled with didactic coursework and hands-on training in Neurovascular Biology, Molecular Biology and Neurotoxicology. The activities outlined in this training application will provide me with training in areas needed for a successfu research career including grantsmanship, project design, and laboratory management. A committee composed of an outstanding team of scientists has been assembled is committed to the candidate and her success and are willing to play an active role in facilitating my growth as a scientist. The University of Wisconsin is a top-tier research institute with numerous NIH-sponsored training programs, core facilities, and outstanding researchers, which makes it an ideal training environment for me to develop as an independent scientist. Ultimately, the training in my research and career development plan, my interactions with my mentors, committee members and consultants, and my outstanding training environment will provide an excellent foundation for a successful academic career at a research intensive academic institution.