ESTABLISHING A ZEBRAFISH MODEL FOR INFECTION-RELATED CONGENITAL DEFECTS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目及 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 微生物致畸剂影响所有人类活产的1-5%，并且是先天性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。母体在妊娠前三个月感染这些病原体会导致严重异常，包括小眼症（小而无功能或功能差的眼睛）。畸形学的机制知之甚少，虽然经胎盘感染的发展中的胚胎参与。 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。斑马鱼发育迅速，外部，并服从遗传，分子和药理学操作，以及直接感染发育中的胚胎。 我们假设发育中的视觉系统通过保守途径对眼睛选择性（遗传）和非选择性（毒素和感染）应激做出反应，导致定型的小眼表型（Kashyap等人，2007年）。 例如，参与眼睛形态发生的基因的实验性敲除导致眼睛尺寸减小，并且严重限制视网膜细胞分化（Stenkamp等人，2002; Shen和Raymond，2004; Rojas-Munoz等人，2005年）。 类似地，用乙醇处理胚胎引起小眼症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微生物畸形学的斑马鱼模型，以进一步验证这一假设。在与COBRE研究人员Gustavo Arrizabalaga和Lee Escherato的合作中，我们将专注于弓形虫和HSV，这两种微生物致畸剂不是物种特异性的。胚胎斑马鱼将在神经形成和器官发生期间暴露于这些感染因子（在8天内的各种暴露时间）。  受精后48小时）。 将评价固定胚胎对眼睛大小和一般发育速度的影响。 我们最初的目标是a）建立允许感染的条件，B）记录任何由此产生的发育异常，以及c）确定对这些微生物致畸剂敏感的任何关键时期。 将胚胎包埋并切片，用于使用原位杂交和免疫细胞化学技术分析眼特异性基因表达，以与其他小眼表型进行比较（参见Kashyap等人，2007年）。未来的研究将使用比较基因分析方法在基因调控水平上验证我们的假设。

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.