ESTABLISHING A ZEBRAFISH MODEL FOR INFECTION-RELATED CONGENITAL DEFECTS

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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.