ESTABLISHING A ZEBRAFISH MODEL FOR INFECTION-RELATED CONGENITAL DEFECTS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 微生物致畸剂影响1-5%的人类活产，是导致先天畸形的主要原因之一。致畸生物包括弓形虫、风疹、巨细胞病毒(CMV)、单纯疱疹病毒(HSV)和梅毒。在怀孕前三个月，母亲感染这些药物会导致严重的异常，包括小眼炎(小而无功能的眼睛或功能不佳的眼睛)。尽管发育中的胚胎经胎盘感染，但其致畸机制仍知之甚少。我们研究计划的一个主要目标是了解视觉系统发育异常产生的机制。我们使用胚胎斑马鱼作为我们研究的模式生物。斑马鱼在外部发育迅速，易受遗传、分子和药物等方面的影响，也能直接感染发育中的胚胎。 我们假设，发育中的视觉系统通过一条保守的途径对眼睛选择性(遗传)和非选择性(毒素和感染)压力做出反应，导致刻板印象的微眼症表型(Kashyap等人，2007年)。例如，实验性地敲除与眼睛形态发生有关的基因会导致眼睛变小，并严重限制视网膜细胞分化(Stenkamp等人，2002；Shenand Raymond，2004；Rojas-Munoz等人，2005)。同样，用乙醇处理胚胎会导致小眼炎和视网膜细胞分化减少(Kashyap等人，2007年)。 我们现在希望建立一个用于微生物畸形学的斑马鱼模型，以进一步验证这一假说。在与Cobre调查人员Gustavo Arrizabalaga和Lee Fortunato的合作下，我们将专注于弓形虫和HSV，这两种微生物畸胎原不是物种特有的。胚胎斑马鱼将在神经形成和器官形成期间(受精后8、48小时内的不同暴露时间)暴露于这些感染源。固定的胚胎将被评估对眼睛大小和一般发育速度的影响。我们最初的目标将是a)确定允许感染的条件，b)记录任何由此导致的发育异常，以及c)确定对这些微生物致畸剂的任何关键敏感期。胚胎将被嵌入和切片，用于使用原位杂交和免疫细胞化学技术分析眼睛特异基因的表达，以便与其他小眼球表型进行比较(见Kashyap等人，2007年)。未来的研究将使用比较基因图谱方法在基因调控水平上测试我们的假设。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Microbial teratogens affect 1-5% of all human live births and are among the leading causes of congenital malformations. Teratogenic organisms include toxoplasma, rubella, cytomeglovirus (CMV), herpes simplex virus (HSV), and syphilis. Maternal infection with these agents during the first trimester of gestation results in severe abnormalities including microphthalmia (small and nonfunctional or poorly functional eyes). The mechanism for teratology is poorly understood, although transplacental infection of the developing embryo is involved. A major goal of our research program is to understand the mechanisms through which developmental abnormalities of the visual system are generated. We use the embryonic zebrafish as a model organism for our studies. The zebrafish develops rapidly and externally, and is amenable to genetic, molecular, and pharmacological manipulations, as well as to direct infection of developing embryos. We hypothesize that the developing visual system responds to both eye-selective (genetic) and non-selective (toxins and infections) stresses through a conserved pathway, resulting in a stereotyped microphthalmic phenotype (Kashyap et al., 2007). For example, experimental knockdown of genes involved in eye morphogenesis results in reduced eye size, and severely limited retinal cell differentiation (Stenkamp et al., 2002; Shen and Raymond, 2004; Rojas-Munoz et al., 2005). Similarly, treatment of embryos with ethanol causes microphthalmia and reduced retinal cell differentiation (Kashyap et al., 2007). We now wish to establish a zebrafish model for microbial teratology, to further test this hypothesis. In collaboration with COBRE investigators Gustavo Arrizabalaga and Lee Fortunato, we will focus on toxoplasma and HSV, two microbial teratogens that are not species-specific. Embryonic zebrafish will be exposed to these infectious agents over the period of neurulation and organogenesis (various exposure times over the 8  48 hours post-fertilization). Fixed embryos will be evaluated for effects on eye size and general pace of development. Our initial goals will be to a) establish the conditions permissive for infection, b) document any resulting developmental abnormalities, and c) identify any critical periods of sensitivity to these microbial teratogens. Embryos will be embedded and sectioned for analysis of eye-specific gene expression using in situ hybridization and immunocytochemical techniques, for comparisons with other microphthalmic phenotypes (see Kashyap et al., 2007). Future studies will test our hypothesis at the level of gene regulation using a comparative gene profiling approach.