From luc to GFP to lux: Evolving an improved zebrafish model for the screening of

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Project Summary. Endocrine disrupting chemicals (EDCs) interfere with the intricate trafficway of hormones that control virtually every organ and system in the human body and elicit consequent developmental and reproductive effects that are of significant human health concern. Major screening programs have been established worldwide to identify and describe the actions of chemicals with endocrine disruptor characteristics, but the process is challenging because of the need to characterize complex EDC modes of action superimposed against an equally complex organismal network of hundreds of circulating hormones that exert widespread tissue/organ-, age-, and sex-specific effects. Despite these intricate whole-body EDC disease manifestations, conventional assays attempting to describe EDC activity are based on in vitro assays that profile chemical mechanisms of action in isolated cell cultures. These assays fail to reveal the tissue- and life stage-specific properties of EDCs and provide few details on critical toxicological endpoints. Conversely, whole-organism in vivo assays are considered more ideally suited for acquiring this information, with the zebrafish serving as a premier model for doing so. Transgenic zebrafish expressing fluorescent reporter proteins have been designed to monitor for EDC exposure effects. However, as the zebrafish ages it accumulates fluorescent pigmentation within its tissue with corresponding loss of optical clarity, making the discrimination of target fluorescent signals practical over only a few days and thus leaving behind far too much information of significant clinical value. Zebrafish transgenics that integrate bioluminescent reporter systems may solve this problem because zebrafish do not display natural bioluminescence and therefore present superior signal-to-noise ratios. We have synthetically optimized the bacterial luciferase (lux) bioluminescent reporter cassette for efficient expression under eukaryotic genetic controls with demonstrated application in mammalian cells and rodent models. We hypothesize that we can use our codon optimization strategy to design a bacterial luciferase that can as well be efficiently expressed in zebrafish and, in association with an amplified estrogen receptor fusion approach, applied as a new in vivo transgenic model for real-time, tissue-specific bioluminescent-based EDC screening across all zebrafish life stages. The specific aims of this research effort are to 1) Express a codon-optimized bacterial luciferase in zebrafish under an amplified estrogen receptor fusion, 2) Validate and characterize EDC exposure response characteristics against a battery of target test compounds, and 3) Investigate tissue- and life stage-specific bioluminescent response profiles to EDC exposures throughout all stages of zebrafish development. This research effort supports the vision of the NIH NIEHS National Toxicology Program to "refine traditional toxicology assays and develop rapid, mechanism-based predictive screens for environmentally induced diseases" and does so in a research environment designed to intellectually stimulate and challenge four undergraduate students.