Journey Upstream Spawns New Research Models

Think outside the cage." That's all it takes to understand why some researchers are moving away from mouse models and diving into transgenic fish research, according to biologist Richard N. Winn of the University of Georgia. Investigators bucking the mammalian tide come from all sectors of biological research, and, in addition to Winn, include National Academy of Sciences (NAS) member Richard B. Setlow, senior biophysicist at Brookhaven National Laboratory; Elwood A. Linney, professor of microbiology at Duke University; and Michael J. Carvan III, assistant scientist, University of Wisconsin, Milwaukee, among others. Courtesy of Brookhaven National LaboratoryRichard Setlow These researchers have been lured, they say, by the research power inherent in fish. These prolific vertebrates are being shown to have a remarkably similar biology to mice, and, as models, fish provide high-quality data. "The objective, always, is to get good statistics and get them rapidly," notes Setlow, "and fish allow you to do that." Fish models offer several distinct advantages over mice. They produce large numbers of offspring more quickly, and the vast species variety allows for enhanced comparison studies. Also, fish cost less to maintain than do mammals, and they attract less attention from animal-rights activists. Although acquiring funding is an upstream journey, investigators using transgenic fish for environmental toxicology and biomedical studies have obtained National Institutes of Health grants. "The environmental health aspects to which these model organisms can contribute is huge," says Carvan, who has developed transgenic zebrafish sensitive to pollutants.1 Linney is also developing biosensors sensitive to toxicants, as well as employing transgenic zebrafish in studies on neural tube defects and the retinoic acid pathway.2 Setlow, who is collaborating with Japanese researchers to explore germ cell mutations from cosmic ray nuclei using medaka fish, is seeking funding to use transgenic medakas developed by Winn as biosensors to test waters on and around Long Island for mutagenicity that may have arisen from use of pesticides and herbicides. And, Winn, who took the plunge as a postdoc at Duke and later at the National Institute of Environmental Health Sciences (NIEHS), is focusing primarily on mutagenesis in medaka and fundulus species.3 Troubled Waters In the late 1960s, Setlow became one of the first researchers to recognize the value of fish. In research at Oak Ridge National Laboratory, he used Amazon molly and Xiphophorus to study ultraviolet damage. "Our colleagues thought we were crazy," he remembers. His work ultimately revealed findings about malignant melanoma,4 but fish as models didn't catch on. The vast majority of researchers viewed fish models with disdain--an attitude that lingers today, Carvan says. When Linney, who established a transgenic mouse facility for the Duke Comprehensive Cancer Center, jumped from mice to zebrafish in the mid-1990s, he recalls getting a similar skeptical reaction, but, like Setlow, he remained steadfast. "I realized I could use zebrafish [for] four-dimensional studies of the developing embryo," he explains. Linney secured funding with relative ease, but most researchers have found that money is difficult to reel in. Some investigators in years past actually resorted to stealth tactics. "A number of colleagues told me they never used the word fish in the title of their grant because it would never be read," Winn confides. "They substituted the word teleosts, [which] sometimes helped." Courtesy of Peter Frey, University of GeorgiaTransgenic mekada The perception of fish, however, is slowly changing. In recent years, the zebrafish has garnered significant NIH backing. When Nobel laureate Christiane Nüsslein-Volhard decided to develop technology for genetically screening embryonic mutant zebrafish, the model gained respectability, Linney says. Researchers at the University of Oregon also "helped keep the [model] alive and open eyes," he acknowledges. Progress is steady. The Sanger Centre, for example, has committed to sequencing the zebrafish genome, which has encouraged Linney's lab to develop "a modest zebrafish microarray for analyzing hundreds to thousands of genes as development occurs or in response to exposure of toxicants." And, in Japan, University of Tokyo researchers are beginning to sequence the medaka genome. Techniques developed for the zebrafish system will transfer to other fish systems in the future, Winn says. But the focus on one species has diverted attention from others, making it harder for people such as Setlow and Winn, who work with other fish model systems, to get the kind of support they need. Other issues also impede development of fish models. Most fish facilities, Winn says, are substandard and researchers must assume responsibility for all care and husbandry, whereas their mouser counterparts often have assistants. Researchers Strategize In meetings at the Southwest Texas State University and at Mount Desert Island Biological Laboratory (MDIBL) in Maine,5 scientists working with fish models planned strategies for boosting their research. "It's a matter of hoisting the profile, and the first step is building community," Winn says. During a keynote talk at the MDIBL meeting in May, Allen Dearry, chief of the Chemical Exposures and Molecular Biology Branch of NIEHS in Research Triangle Park, N.C., emphasized the need for better marketing and for developing advocates for different species. "I think [researchers] have shown that there are a number of potential models that have their own strengths," Dearry told The Scientist later. "What might be considered are more collaborative projects between different labs and investigators, ... [which] could well be more successful than individual efforts because they have the potential to lead to larger contributions and greater advances." Winn, who has at least six collaborations under way and heads a state-of-the-art transgenic fish facility, agrees. "Transgenic fish research is really helping to change the approach to research overall because of the need for multidisciplinary input." A critical next step, Carvan adds, is the establishment of "a series of virtual centers backed up by physical centers to handle bioinformatics, genomic resource needs, or transgenic needs--a system that would allow researchers from different universities to interact." Smoother Seas In the future, says Setlow, fish transgenics will be "a big area where a lot of people can work easily" and will become "increasingly important." Fish offer researchers something remarkable, he adds, "the opportunity to study the critical interval between conception and birth when mutations as a function of age tend to be the greatest." Courtesy of Elwood A. LinneyTransgenic zebrafish embryo Researchers are already developing multicolored fish with color reporter genes so that certain elements of the embryo will light up blue, green, or red, Linney adds. "And, in the next five years, techniques such as fluorescent resonance energy transfer, which allows you to change the color of fluorescence when two molecules get within molecular distance of each other, will be used to watch molecules talking at the membrane and within individual cells." On the environmental front, with global water supplies becoming dangerously polluted and industry's need to efficiently test new compounds, fish biosensors will soon be in high demand, Carvan says. Linney predicts: "The next generation will have to know something about fish." In the meantime, the fish researchers carry on. "One real beauty of fish is that they allow your research questions to be the drivers and that is so important," says Winn. "As the field progresses, people will see the evolution of thinking 'outside the cage,' and remember that some of our greatest enlightenment comes from looking at things from another angle." Setlow concludes: "People just have to realize that fish are reputable, and that there are great similarities to mammalian systems. The old biologists always would say, 'ontogeny recapitulates phylogeny.' [He pauses for reflection] Besides, why do people work with worms? And what's so great about a mouse that isn't so great about a fish?" A.J.S. Rayl (ajsrayl@loop.com) is a contributing editor for The Scientist. References 1. M.J. Carvan III et al., "Transgenic zebrafish as sentinels for aquatic pollution," Annals of the New York Academy of Science, 919:133-47, 2000. 2. A. Perez-Edwards et al., "Retinoic acid-mediated gene expression in transgenic reporter zebrafish," Developmental Biology, 229: 89-101, 2000. 3. R.N. Winn et al., "Detection of mutations in transgenic fish carrying a bacteriophage lcII transgene target," Proceedings of the National Academy of Sciences (PNAS), 97:12655-60, 2000. 4. R.B. Setlow, et al., "Wavelengths effective in malignant melanoma," PNAS, 90:6666-70, 1993. 5. A.J.S. Rayl, "How to create a successful fish tale?" The Scientist, 15[16]:1, Aug. 20, 2001.

Journey Upstream Spawns New Research Models

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