How to Create a Successful Fish Tale?

More than 80 percent of the planet's living organisms exist only in aquatic ecosystems. Some may harbor secrets to human origins, and clues, treatments--perhaps even cures--for human disease. Some are critical bioindicators that portend the health of the biosphere. Yet, overall, scientists know little about the biochemical processes of these life forms. The vast, rich knowledge within the oceans and freshwater systems on Earth remains virtually untapped, because in the world of biological research, mammals rule. A pool of aquatic researchers convened recently at the Mount Desert Island Biological Laboratory (MDIBL) in Salisbury Cove, Maine, to figure out how to change that. These investigators shared research findings, successes, failures and insights, and participated in workshops to devise strategies on how to raise their collective profile and put the water world on the research map. "We've been dealing with a very human-mouse centered culture of science," says MDIBL director John N. Forrest Jr., professor of internal medicine at Yale University. "Yet the sea is a place of enormous diversity, and it holds the potential for discovery of all kinds of things--and this is the beginning of our pitch." During approximately 150 years of cell biology history, only a handful of marine cell lines have been established, and the government has earnestly funded only one transgenic species of fish. It's ironic, considering that long-time director of the National Institutes of Health, James A. Shannon, who oversaw a major growth period of the agency during 1955-68, spent many summers at MDIBL conducting research on marine species. Despite discoveries made by Shannon and others over the years, however, "nobody really viewed this research, generally, as all that important until fairly recently," says John P. Wise, assistant professor of epidemiology/public health, Yale University and MDIBL. Within the last decade, however, more scientists have come to realize just how far-reaching the flow of knowledge from the development of transgenic fish models and teleost and marine mammal cell lines may be. Applications are already emerging in aquaculture, biomedicine, biotechnology, ecotoxicology, environmental health, fundamental physiology, and genetics, among other specialized fields. If aquatic research can secure serious support, these researchers avow the data will come in deluges. Moreover, in the grand global scheme, an increasing number of scientists have come to view the oceans as "the lifeblood of the planet," as Thomas J. Goodwin, manager, biotechnology flight definition, NASA, puts it. "The secrets of the seas are critical to human as well as marine mammal survival." Beyond the Zebrafish The zebrafish, to date, is the only fish to have landed solid governmental funding. Thomas T. Chen, professor-director, University of Connecticut Biotech Center, maintains, however, "Fundulus, sheepshead minnow, three-spine stickleback, and other fish species [which, unlike the zebrafish, still exist in the wild] are highly desirable models for biomedical and environmental research, as well as experimental models for basic research," and in many cases are highly superior to zebrafish. Moreover, he adds, "The transgenic technology has great potential for biotech applications, including increased somatic growth rate in aquaculture fish, and the ability to breed disease-free fish and value-added fish." With more fisheries drying up, and various other species in the wild succumbing to toxins, aquaculture is now being viewed as a critical food production source in coming years. Transgenic fishes also promise to transform the landscape of research. "When you apply the use of fish as alternative nonmammalian models, you can have the questions drive the science, rather than being married to established models," contends Richard N. Winn, associate professor, University of Georgia. "A good example has been developed by the Japanese in the form of a mutant, transparent strain of medaka. You can see whole adult live fish with all their internal organs. In particular, they developed a GFP fish that fluoresces green only in the testes or the ovaries. This would allow you to put the fish under mild anesthesia, then under a microscope to actually observe those various organs. You could never do this in a mouse. It's the most bizarre and amazing thing, and it means you could actually use this critter for cancer testing." Intriguingly too, as science advances in genetics, "we are beginning to see that there are a lot more similarities in fish with other models than differences," reveals Winn. Adds Forrest: "The greatest power of genes from the sea right now--from sharks, killifish, skates--is that they are so much older than mammalian genes, and that can give us big clues as to the function of genes in man." Some significant work in aquatic creatures has been done. Studies of the shark rectal gland--to which Forrest has made notable contributions--has led to some fundamental discoveries in cystic fibrosis and salt transport across epithelial cells.1-3 But this model and others beg to be more fully developed to define the true extent of their importance. "The need for continuous cell lines is, really, an issue of simple environmental stewardship," Wise expounds for The Scientist. "We really don't know what's going on in our rivers and oceans. Fish and marine mammal cell lines are needed now, he continues, to maintain healthy populations. "The marine mammals--dolphins, seals, whales, and others--are bioindicators of the health of the oceans ... [and] critical for coastal economies. Right now, many of those populations are in crisis. A population of stellar sea lions in Alaska has absolutely plummeted over the last 10, 20 years and there is no clear explanation why. And, right now, we don't have any adequate models to test what in the water could be causing the problem." Courtesy of MDIBLAquatic researchers gathered together at MIDBL to discuss strategies on raising more interest in marine science Increasing Visibility As a community, these researchers know that the first thing they must do is educate the rest of the mammalian-oriented scientific community to garner advocates, increase their visibility, and secure more solid funding. "I have been astounded with the lack of the breadth of knowledge of some scientists," says Winn. "For example, I just returned from a meeting in Japan and a number of researchers pointed out that ... mammalian toxicologists have often asked whether or not fish might be valuable for lung cancer research--and they found themselves constantly having to say, 'Uhh, fish don't have lungs.'" (With very few exceptions, Winn adds later, "such as the lung fish.") Another part of that education, as Winn sees it, is to convey that the diffusion among those working in fish models is a good thing, "a strength," he contends. "While it may be more efficient to focus on one model, it may have less vision. Recognizing that, we must broaden the view for a lot of our own colleagues and scientists and say, 'Look there's a lot of different ways we can approach a number of questions, let's not rely on one.'" The journey of a thousand miles begins, they say, with the hoisting of the sails, and by the end of the MDIBL gathering, the course had been charted with a 'sea map' leading straight to Bethesda, Md., and Washington, D.C. Their chart calls for a water world resource to be built in three phases, beginning with virtual repositories in the form of Web sites, one devoted to transgenics and one to cell lines. Next, once funding is acquired, they hope to establish two national centers or physical repositories--one for cell lines and one for transgenics--both of which would incorporate and coordinate with the Web repositories. Such national repositories would, the group decided, be established in a global effort. "To do this effectively, we have to establish international links to groups doing similar things in Japan, Europe, and elsewhere to ensure there is connectivity," says John J. Stegeman, senior scientist, Woods Hole Oceanographic Institution, Woods Hole, Mass. "Whatever we develop here should be closely linked and tied to efforts overseas as well." He adds, because the data and materials they are seeking to generate would be used in a variety of ways in numerous fields, that funding should be sought from, in addition to the NIH, the National Oceanic and Atmospheric Administration, National Science Foundation, Environmental Protection Agency, Food and Drug Administration, and other agencies. In the weeks following the MDIBL conference crosstalk flew and collaborative teams began to form. Some of the investigators are in discussion on joint projects, a book on fish cell culture is in the works, and virtual repositories for both aquatic cell lines and transgenics are in the developmental stages. A marine mammal working group has banded together and is already forming a network of field laboratories coast to coast to collect tissue from stranded or found animals and develop the primary cultures, then send them to MDIBL to be immortalized. The completed cell lines will be stored, in the immediate future, at Mystic Marine Life Aquarium, Mystic, Conn., which will make them available to other researchers. Once they establish the network and begin the process with a couple of animals, they will go to Congress to seek funding for an official national repository. "I don't see any point in going slowly," Wise says, "We're aiming to deliver the plan in Washington next year." A.J.S. Rayl (ajsrayl@loop.com) is a contributing editor for The Scientist. References 1. S.G. Aller et al., "Cloning, characterization, and functional expression of a CNP receptor regulating CFTR in the shark rectal gland," American Journal of Physiology-Cell Physiology, 276:C442-9, 1999. 2. J.N. Forrest Jr., "Cellular and molecular biology of chloride secretion in the shark rectal gland: regulation by adenosine receptors," Kidney International, 49:1557-62, 1996. 3. R. Greger et al., "The cellular mechanisms of CL-secretion induced by c-type natriuretic peptide (CNP). Experiments on isolated in vitro perfused rectal gland tubules of Squalus acanthias," Pflugers Archiv--European Journal of Physiology, 438[1]:15-22, 1999. Oceanic Haven Houses First Center of Its Kind The Mount Desert Island Biological Laboratory (MDIBL) borders the Atlantic Ocean just outside Maine's Acadia National Park, not far from the popular summer resort town of Bar Harbor. Established 102 years go in the wake of the Darwinian revolution, it is one of the oldest surviving independent marine stations in the country. Over the decades, the laboratory has drawn world-class researchers to its shores and garnered a reputation for its scientific and educational programs. Two years ago MDIBL marked another marine milestone by opening the Center for Marine Functional Genomic Studies, the first sequencing center in the world dedicated to marine organisms. Although MDIBL has been a summer-only institute for almost its entire history, this year full-time research scientists will be brought on board to work year-round. MDIBL is currently looking for research scientists in the fields of toxicology/toxicogenomics and cell biology/physiology. Courtesy of Mount Desert Island Biological LaboratoryThe Mount Desert Island Biological Laboratory's conference center Interestingly, the lab and the concept behind it first began in the southern part of state, in Harpswell as a summer school for Tufts University biology students. Then as Acadia National Park on Mount Desert Island was established, a group of businessmen from Bar Harbor purchased tracts of land to create a place for a marine station. In 1921, MDIBL opened its doors. "The idea was to set aside this area for people of science to get away from the hustle and bustle and be able to observe and study nature," says lab director John N. Forrest Jr. of Yale University. MDIBL's contribution to the biology of the 20th century is noteworthy. "It was James A. Shannon, who former President Franklin D. Roosevelt called to [head the National Institutes of Health], and E.K. Marshall and Homer Smith,1 who investigated renal physiology up here and discovered how the kidney and the gill regulated the salt concentration of cells in the body," informs Forrest. "So they came up here in part to understand the physiology of things like salt-sodium, potassium chloride, and calcium, and, with Robert Berliner and others, they also studied the evolution of life from the sea to land through the function of these specialized organs." Other notable discoveries have emerged from MDIBL. In 1959, J. Wendell Burger discovered the function of the shark rectal gland, a specialized gland of one cell type exists in the intestine and functions to pump out salt.2,3 Says Forrest, "It now turns out that the shark salt gland is a phenomenal model for the disease cystic fibrosis, which is a disease of a chloride abnormality, where the lungs and airways don't secrete enough chloride.4 There is a similar gland in the nose of seabirds--both these glands were discovered at this lab." Squalamine--"one of the secrets the shark had as a way of getting around infections or preventing cancer," as Forrest describes it--was found in sharks studied at MDIBL and developed into an antibiotic.5 Currently, squalamine, which "so far [has] only been found in the shark," is being tested in cancer trials. At the Center for Marine Genomic Studies other scientists will be sequencing some model organs, including the shark rectal gland. "Doing the whole genome is prohibitive at this point, so we are doing express sequence tags of the maybe 15,000 genes in the shark rectal gland, for example, and the gill," says Forrest. The genes will be banked in the center's database and made available for those who want to study them. --A.J.S. Rayl References 1. H.W. Smith, "The absorption and excretion of water and salts by the elasmo branch fistus," American Journal of Physiology, 98:296-310, 1931. 2. J.W. Burger and W.N. Hess, "Function of the rectal gland in the spiny dogfish," Science, 131:670-1, 1960. 3. J.W. Burger, "Roles of the rectal gland and of the kidneys in salt and water excretion in the spiny dogfish," Physical Zoology, 38:191-6, 1965. 4. J. Marshall et al., "Identification and localization of a dogfish homologue of human cystic fibrosis transmembrane conductance regulator," Journal of Biological Chemistry, 33:22749-54, 1991. 5. M. Zasloff et al., "Megainin-like anti-bacterial properties of tissue extracts from Squalus acanthias myxine glutinosa," Bulletin MDIBL, 29:137-8, 1990. Aquatic Research Spans Spectrum The talks and poster presentations given during the recent gathering at Mount Desert Island Biological Laboratory (MDIBL) in Salisbury Cove, Maine, revealed the spectrum of recent and ongoing investigations in the ponds of fish transgenics and aquatic cell lines, and established the relevance and need for well-funded research. Mammals may rule in the world of biological research, but fish are the most diverse class of vertebrates, with approximately 20,000 different species occupying all aquatic niches. Despite the fact that transgenic fish were introduced in the 1980s and have been found to be excellent models for disease, as well as offering investigators tremendous opportunities in the study of carcinogenesis, mutagenesis, and endocrine disruption, precious few lineages are available, and they are still not being produced routinely. Among the presentations: Robin A. Wallace, professor emeritus, fisheries and aquatic sciences at the University of Florida, reviewed the areas of research relevant to transgenic fish, from aquaculture to biomedicine to nutrition, and discussed the transgenic assets of various fish species. He revealed that Fundulus heteroclitus (Fundulus), Cyprinodon variegates (sheepshead minnow), Gasterosteus aculeatus (threespine stickleback), and Oryzias latipes (medaka) show great promise as transgenic models. Richard N. Winn, associate professor, University of Georgia, honed in on the myths and realities in the world of fish transgenics, noting that although gene transfer is "easy," establishing and maintaining lineages remains "the hard part." Those in the field must continue to work to get fish accepted, he stressed, as respectable models in research. Thomas T. Chen, professor-director, University of Connecticut Biotech Center, concentrated on the latest advances in methodologies and production techniques. He and other researchers are working now to develop fish that produce antibodies, which will alleviate concern about infections in aquaculture farms. Michael J. Carvan III, assistant scientist, University of Wisconsin, Milwaukee, shared recent findings in developing pollution inducible transgenes in zebrafish. He stressed the need for "huge attention to detail" and discussed the numerous reasons a variety of aquatic models needs to be developed. Zebrafish, he noted, "have really tiny livers," and that requires researchers to be tremendously focused in the midst of the "blood bath" that inevitably occurs with examination. John J. Stegeman, senior scientist and biology department chair at Woods Hole Oceanographic Institution (WHOI), talked about the potential for Fundulus heteroclitus as a model in fundamental and environmental toxicology, noting the contribution of WHOI associate scientist Mark E. Hahn. In comparing and contrasting assets, Stegeman showed why fundulus is far superior in terms of environmental relevance than zebrafish. Stegeman zeroed in on the big problem in the realm of fish transgenics: "What is needed to develop fundulus or any other model is what the zebrafish had: an advocate." University of Minnesota professor Perry B. Hackett, president of Discovery Genomics Inc., St. Paul, Minn., brought the group up to speed on two new genetic tools his lab has produced for investigating gene expression in fish-a vertebrate transposon system, Sleeping Beauty (SB), for introducing new genetic material into chromosomes; and morpholinos, which inhibit translation of RNA, and have shown, he says, "an unparalleled anti-sense activity in zebrafish embryos." John F. Lechner, director, oncology program, Bayer Diagnostics in Berkeley, Calif., devoted his talk to transferring the lessons learned from traditional mammalian cell culture science to aquatic systems. Among the considerations for cell culture that aquatic researchers should take into consideration are: toxicity, tissue preparation, attachment, nutrients, physiological factors, subculturing, short cuts, and differentiation. Niels C. Bols, professor of biology, University of Waterloo, gave a highly animated talk on the trials, tribulations, and successes in establishing long-term hemopoietic cell cultures and developing fish cell lines. The study of hemopoiesis (formation of blood cells) in fish is important, he noted, to develop methods for monitoring the health of fish for aquaculture and ecotoxicology and to exploit the unique features of different fish species. In spite of the power of fish to serve as a warning of the impact of toxicants on the aquatic environment as well as the potential impacts on human health, he maintained, "you really need some repository for this business to grow." Rita Anne Garrick, associate professor, Fordham University, and Woods Hole, chronicled her work with the eel, Anguilla rostrata, which offers, she notes, an alluring characteristic in its size, giving ONE "the ability to manipulate the animal." In discussing the isolating and culturing of capillary endothelial cells, she emphasized the value of the cell system to correlate with the intact organ and described in detail how she managed to obtain the cells to use in this way. David M. Barnes, director, National Stem Cell Resource for American Type Culture Collection (ATCC), reviewed the advantages and limitations fish cell culture. He concluded by reiterating the reality that "you just can't put these models in mammalian cell culture and get what you want." With regard to marine mammals, Thomas J. Goodwin, manager, biotechnology flight definition, NASA, shared his experience in the collection of bowhead cells from the annual subsistence hunts by the Inuit in Alaska, and he discussed the immortalization process of those cells in two and three-dimensional cultures. Although indicators have previously pointed to whales having an ability to tolerate carcinogenic concentrations of toxins and metals, the bowhead, for one, is now starting to show signs of disease. Moreover, he pointed out that the species with the most similar metallothinine protein sequence to the bowhead-other than human-is bovine and wondered if the diseases now prevalent in Europe may be transmittable to the marine environment and to the bowhead. In a talk that really got the creative-investigative juices flowing, Shawn E. Holt, assistant professor, Medical College of Virginia Commonwealth University, enlightened the audience on the viability and value of telomerase-what may be "the molecular version of the Fountain of Youth"- in the creation of both cell lines and transgenics. Among the poster session on marine mammals, John P. Wise, assistant professor, Yale University and MDIBL, and Bobby L. Middlebrooks, professor of biology, University of Southern Mississippi, offered up reviews of their work in the establishment of cell lines from a range of cetaceans, including the beluga whale and various species of dolphins, which Middlebrooks found "demonstrated broad susceptibility to viruses" from a number of viral families. -A.J.S. Rayl References for Further Reading Y.L. Tang et al., "Influence of environmental salinity on messenger RNA levels of growth hormone, prolactin, and somatolactin in pituitary of the channel catfish (Ictalurus punctatus)," Marine Biotechnology, 3:205-17, May-June, 2001. R.N. Winn, "Transgenic fish models in environmental toxicology," ILAR Journal, National Academy of Sciences, Special Issue: Fish Models in Biomedical Research, 42:4, 2001. S.M. Bello et al., "Acquired resistance to aryl hydrocarbon receptor agonists in a population of Fundulus heteroclitus from a marine Superfund site: In vivo and in vitro studies on the induction of xenobiotic-metabolizing enzymes," Toxicological Sciences, 60:77-91, 2001. M.J. Carvan III et al., "Transgenic zebrafish as sentinels for aquatic pollution," Annals of the New York Academy of Sciences, 919:133-47, 2000. M.J. Carvan III et al., "Activation of transcription factors in zebrafish cell cultures by environmental pollutants," Archives of Biochemistry and Biophysics, 376:320-327, 2000. R.A. Garrick, "Isolation and culture of capillary endothelial cells from the eel, Anguilla rostrata", Microvascular Research, 59: 377-85, 2000. T.J. Goodwin et al, "A three-dimensional model for assessment of in vitro toxicity in Balaena mysticetus renal tissue," Journal of Applied Physiology, 89:2508-17, 2000. M.F. Oleksiak et al., "Identification, functional characterization and regulation of a new cytochrome P450 subfamily, the CYP2Ns," Journal of Biological Chemistry, 275: 2312-2321, 2000. R.N. Winn et al., "Detection of mutations in transgenic fish carrying a bacteriophage lcII transgene target," Proceedings of the National Academy of Sciences, 97:12655-60, 2000. L. Caldovic et al., "Position-independent expression of transgenes in zebrafish," Transgenic Research, 8:321-34, 1999. L.I. Gonzalez-Villasenor, T.T. Chen, "Antibodies for growth hormone and prolactin using multiple antigen peptide immunogens," Marine Biotechnology, 1:211-20, S.I. Karchner et al., "Identification and functional characterization of two highly divergent aryl hydrocarbon receptors (AHR1 and AHR2) in the teleost Fundulus heteroclitus. Evidence for a novel subfamily of ligand-binding basic helix-loop-helix Per-ARNT-Sim (bHLH-PAS) factors. Journal of Biological Chemistry, 274: 33814-24, 1999. C.P. Morales et al., "Absence of cancer-associated changes in human fibroblasts immortalized with telomerase," Nature Genetics, 21:55-8,1999. A.G. Bodnar et al., "Extension of life-span by introduction of telomerase in normal human cells," Science, 279:349-52, 1998. H.G. Morrison et al., "Molecular cloning of CYP1A from the estuarine fish Fundulus heteroclitus and phylogenetic analysis of CYP1A genes: Update with new sequences," Comparative Biochemistry and Physiology, 121C: 231-40, 1998. Z. Ivics et al., "Molecular reconstruction of Sleeping Beauty, a Tc1-like transposon from fish, and its transposition in human cells," Cell, 91: 501-10, 1997.

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