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MARINE ORGANISMS AS MODEL SYSTEMS

Scientific Questions

The ocean is a source of organisms that are used as model systems to address basic biology questions (Powers, 1989), some yet to be defined. Although automated identification of many marine taxa and assessment of functional diversity appears achievable (see Chapter 3), determination of the physiological status of marine organisms and the development of biological sensors is presently limited by the lack of fundamental information on the physiology, cell biology, and molecular biology of these organisms. In addition, the physiological basis for biological processes, such as recruitment, will require an understanding of the physiological and molecular basis for development, reproduction, nutrition, growth, speciation, and other fundamental information that is the foundation of the phenomenon that biological oceanographers wish to understand.

Marine organisms are used as models to address fundamental questions about the structure and function of cells, subcellular structures, organelles, organ systems, and whole organisms. A variety of molecular approaches can be used to elucidate modes of regulation and address questions about cell receptors, signal transduction, mechanisms of biosynthesis, and cellular transport. Some examples of representative areas that are already being actively analyzed at the molecular and biochemical levels are listed in Table 4-1 to illustrate the diverse, and potentially invaluable, biological systems for which marine organisms provide unique research advantages. Many more examples could be cited, and the list will become vastly expanded as our knowledge increases.



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MOLECULAR BIOLOGY IN MARINE SCIENCE: SCIENTIFIC QUESTIONS, TECHNOLOGICAL APPROACHES, AND PRACTICAL IMPLICATIONS 4 MARINE ORGANISMS AS MODEL SYSTEMS Scientific Questions The ocean is a source of organisms that are used as model systems to address basic biology questions (Powers, 1989), some yet to be defined. Although automated identification of many marine taxa and assessment of functional diversity appears achievable (see Chapter 3), determination of the physiological status of marine organisms and the development of biological sensors is presently limited by the lack of fundamental information on the physiology, cell biology, and molecular biology of these organisms. In addition, the physiological basis for biological processes, such as recruitment, will require an understanding of the physiological and molecular basis for development, reproduction, nutrition, growth, speciation, and other fundamental information that is the foundation of the phenomenon that biological oceanographers wish to understand. Marine organisms are used as models to address fundamental questions about the structure and function of cells, subcellular structures, organelles, organ systems, and whole organisms. A variety of molecular approaches can be used to elucidate modes of regulation and address questions about cell receptors, signal transduction, mechanisms of biosynthesis, and cellular transport. Some examples of representative areas that are already being actively analyzed at the molecular and biochemical levels are listed in Table 4-1 to illustrate the diverse, and potentially invaluable, biological systems for which marine organisms provide unique research advantages. Many more examples could be cited, and the list will become vastly expanded as our knowledge increases.

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MOLECULAR BIOLOGY IN MARINE SCIENCE: SCIENTIFIC QUESTIONS, TECHNOLOGICAL APPROACHES, AND PRACTICAL IMPLICATIONS Major discoveries of fundamental biological processes—many important to human health and medical research—have come from studies of marine organisms. These include the discovery of cellular immunity, which was first recognized in starfish; discovery of the cellular and biochemical mechanisms controlling fertilization, which have been extensively studied in the sea urchin, abalone, and several other marine organisms; discovery of the mechanisms of nerve cell transmission for which giant cells of the squid have been particularly useful; discoveries of the mechanisms of nerve-to-nerve synaptic transmission, studied in the electric fish; insights into the cellular and molecular mechanisms responsible for learning and memory, for which certain marine snails present great experimental advantages; discoveries relating to neurosecretion and neuroendocrine regulation of reproduction, cardiac physiology, and daily rhythms of neuronal and physiological activities; and fundamental analysis of cell interaction, morphogenesis, and the molecular mechanisms of gene regulation in developing embryos. In this latter area sea urchin embryos have been a major experimental model for over a century, and ascidian, molluscan, and other embryos have been studied extensively as well. Table 4-1. Examples of Biological Systems for Which Marine Animals Provide Unique Opportunities for Discovery. ORGANISM BIOLOGICAL SYSTEM Ctenophores Biological-light producing system Squid Electrical impulse transmission system in nerves Sea snails Networks linking neuronal pathways, hormones, and behavior Sea urchin embryos Gene regulation systems Clam, sea urchin, and tunicate eggs Cell division and cytoskeletal control systems Abalone and sea urchin gametes Sperm-egg recognition systems Sharks, hagfish, tunicates, and sea urchins Novel and diverse immune systems that control bacteria and fungi Abalone and sea urchin larvae Cellular response and induction systems responding to environmental signals Molluscan shell glands, echinoderm skeletogenic tissues Cellular and molecular biofabrication systems for unique protein-mineral materials

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MOLECULAR BIOLOGY IN MARINE SCIENCE: SCIENTIFIC QUESTIONS, TECHNOLOGICAL APPROACHES, AND PRACTICAL IMPLICATIONS There is a need to understand the basic mechanisms by which genes control biological functions. The reproduction, development, differentiation, growth, recruitment, and life history patterns of all organisms are controlled by genes. The genes that encode the properties of cells and organisms are themselves controlled by the products of regulatory genes, some of which are sensitive to external environmental cues, others of which are activated by intercellular signals, while others operate according to predetermined genetic programs. The complexities of gene regulation and the consequences for biological form and function have attracted the attention of molecular biologists for the past decade. Although major advances have been made in our understanding, much remains obscure on how genes determine biological functions, how they generate biological structures and materials, and how they control acclimation and adaptation. Among the most serious and general practical failures that will occur if our level of basic knowledge about molecular marine biology is not dramatically improved, are the following: (1) biotechnology research and development will miss opportunities (as noted below in Practical Implications); (2) we will be unable to predict the adaptive capability of marine species of economic and ecological importance to short-term changes in their environment or to global climate change; and (3) we will be unable to achieve a mechanistic understanding of the reproductive processes that control the population sizes of marine organisms or of the factors underlying the temporal and spatial variability of marine organisms. Without this kind of understanding, control of the ecological interactions of important marine species will never become possible. Signal Molecules and Genes Controlling Reproduction, Development, and Growth The reproduction, growth, and recruitment of marine organisms are controlled by molecular signals--molecular response mechanisms that can now be identified with the tools of molecular biology. It is possible, for the first time, to reliably control these processes in several valuable species, to attain improved efficiency and enhanced yields in mariculture, and to understand and predict variations in recruitment of valuable ocean stocks. The signals controlling these processes include molecules from the environment, molecules produced by other organisms, and molecules produced internally, that is, within the organism. A major goal of molecular marine biology is the identification of these signal molecules and the genes that control their structures, their biosynthesis, their recognition, and the responses of these signals. This is important because the signaling molecules and their receptors are among the most highly conserved proteins, and thus discovery of new signaling molecules and receptors in marine organisms will likely lead to the discovery of homologous molecules in vertebrates (e.g., Baxter and Morse, 1992). The point here is that the enormous diversity of

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MOLECULAR BIOLOGY IN MARINE SCIENCE: SCIENTIFIC QUESTIONS, TECHNOLOGICAL APPROACHES, AND PRACTICAL IMPLICATIONS marine animal life offers a rich variety of opportunities for discovery. In addition, harnessing these signals, their receptors, their signal transducers, and the genes that encode them would have immediate practical applications in aquaculture, and advances in this area already have increased the possibilities of this industry in the United States. The near-term payoffs, however valuable, will constitute only a minor preview of the usefulness of this knowledge a few years from now, when much more sophisticated mariculture and biotechnological applications will become possible. This focus is reflected in the major goals of the new international programs on reproductive biology in aquaculture recently adopted by the International Union of Biological Scientists. Neurobiology Marine organisms play a central role in experimental research aimed at understanding cellular and molecular aspects of the central nervous system. Historically, the nerve impulse or action potential was discovered in a marine mollusc, the squid. Two examples serve to illustrate the importance of marine organisms in neuroscience research. First, the marine snail Aplysia is widely used as a preparation for investigating the cellular regulation of behavior, including learning and memory. The neurons in Aplysia can be up to one millimeter in diameter, large enough to see with the naked eye. This special property allowed investigators to correlate the activity of individual nerve cells with specific behavioral patterns involved in locomotion, feeding, defensive movements, and reproduction. The neuronal circuitry--that is, how the cells are connected and how they communicate with each other--has been defined. When the animal experiences various stimuli the behavior is changed. The experience is encoded in the neuronal circuit and gives rise to a modified behavior. Since the circuitry is known, a cellular analysis of the behavioral modification has been possible to achieve experimentally. These studies have shown that the amount of chemical messenger released from a given cell and detected by a second cell is increased. This makes the connection between the cells stronger and is currently a fundamental cellular mechanism of memory (see review of Jung and Scheller, 1991). A second group of marine organisms of importance to neuroscience research is the marine ray (primitive fish). These animals have been an electromotor system that is used as a defensive reflex or to shock prey prior to feeding. Nerve cells communicate by releasing chemicals called neurotransmitters from a specialized structure called a synapse. The density of synapses in the marine ray electric organ is about 100 times the concentration in human muscle and allowed the purification of a large variety of the molecules that comprise the synapse. These

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MOLECULAR BIOLOGY IN MARINE SCIENCE: SCIENTIFIC QUESTIONS, TECHNOLOGICAL APPROACHES, AND PRACTICAL IMPLICATIONS molecules include the receptors for the neurotransmitter, the acetylcholine receptor (AChR). The AChR is currently the best-understood neurotransmitter receptor; it acts by opening a channel in the cell membrane so that ions can rapidly enter the cell when the signaling molecule (i.e., acetylcholine) binds to the receptor (reviewed by Karlin, 1991). This is a major mechanism of cellular response to external signals in the nervous system of all animals, including humans. Marine animals are valuable for neuroscience research aimed at understanding diseases as diverse as learning disabilities in children to Alzheimer's disease in the aged. Differentiation and Pattern Formation Morphological development is generated by regulated expression of genes early in the life cycle. It is these gene regulatory programs that must have arisen in evolution to account for the appearance of new life forms, by change or reassembly of preexisting regulatory programs that must have arisen in evolution to account for the appearance of new life forms, by change or reassembly of preexisting regulatory circuitry. These mechanisms are at the very heart of speciation in the ocean and reflect the evolutionary adaptation of higher taxonomic groups to changes in marine environmental parameters. That is, the biological and morphological properties of organisms are controlled by gene regulatory programs, and it is the different properties of organisms that enable them to adapt evolutionarily. Therefore, study of the molecular biology of gene regulation in differentiation intersects with the study of ecological adaptation and the evolutionary origin of organismic novelty. The union of these disciplines will require advanced molecular techniques now being developed that will enable identification, isolation, and analysis of key genes controlling cell type, pattern formation, and construction of adaptive traits, including in particular the isolation of regulatory gene products that control expression of these genes. The basic strategy has already been shown in the analysis of regulatory systems involved in the development of Drosophila and sea urchin embryos. Evidence obtained in these and other systems has demonstrated that evolution has functioned conservatively in utilizing the same active domains of genes and proteins in many different contexts. This has immediate and essential practical significance: basic discoveries of active regulatory molecules made in lower organisms, including invertebrates, often lead directly to the discovery of hitherto unknown homologous regulatory genes and proteins that are essential for normal vertebrate development and organ function. Understanding development means understanding how multicellular organisms are progressively created through expression of the genetic programs carried in the egg and sperm nuclei (Davidson, 1990, 1991). Encoded in the genomic DNA is sequence information for all the proteins that endow the

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MOLECULAR BIOLOGY IN MARINE SCIENCE: SCIENTIFIC QUESTIONS, TECHNOLOGICAL APPROACHES, AND PRACTICAL IMPLICATIONS differentiated cells of the organism with their special functional properties. The genome also contains the crucial regulatory information that ultimately ensures that the appropriate proteins are expressed in the appropriate spatial and temporal domains of the organism. Developmental gene regulation has been intensely studied in the sea urchin embryo, because of the practical advantages afforded by the availability of immense numbers of synchronously developing embryos; their permeability to radioisotopes; the ease of exogenous gene transfer into these eggs; accessibility to a variety of optical, cytological, biochemical, and molecular technologies; and because of their particularly simple, straightforward, early developmental process. Research on sea urchins demonstrates the type of approaches that could be extended to other organisms. To achieve a mechanistic understanding of the developmental process is a fundamental goal of life sciences. Enough is known to perceive what will be required: isolation and characterization of marker genes that represent the various differentiated cell states of the developing organism; analysis of their gene regulatory systems; cloning and characterization of the regulatory proteins that determine their differential functions; and knowledge of the ligand-receptor interactions and signal transduction pathways by which the genetic and metabolic activities of each embryonic cell are related to those of adjacent cells. Knowledge of these problems is elemental to understanding the basic biological character of all metazoan organisms, plant and animal, as they are all the product of developmental processes. Marine organisms have an essential role to play in the experimental endeavor to comprehend the mechanism of development. The ramifications of this endeavor extend all the way from basic enlightenment—how we get to be what we are—to high technology. First, marine organisms offer unique practical experimental advantages for the molecular study of development, as very briefly indicated above for what is currently the best-known marine example, the sea urchin. That is, by studying these organisms we can learn more, faster, and at immensely less cost, than we can by studying vertebrates, for instance. From one mouse, 50 to 80 eggs can be recovered, and these eggs can achieve only a very primitive state of development in vitro, that is, before implantation into the wall of the uterus. After this, it is technically very difficult (though of course not impossible) to have experimental access to them. One Strongylocentrotus purpuratus (the common California purple sea urchin) female will produce 20 million to 30 million eggs (of about the same dimensions as a mouse egg), which carry out complete development in vitro in the lab in about 72 hours. To isolate the rare molecules, present at only a few hundred to a few thousand molecules per nucleus, that regulate genes, it is immensely important to have available adequate quantities of developing material so that biochemical methods may be used. In addition, there are numerous negative attributes associated with exclusive reliance on mammalian

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MOLECULAR BIOLOGY IN MARINE SCIENCE: SCIENTIFIC QUESTIONS, TECHNOLOGICAL APPROACHES, AND PRACTICAL IMPLICATIONS research models, ranging from their very high relative expense, to social attitudes. Second, marine metazoa offer by far the greatest biological diversity on the face of the earth. Diversity in developmental process underlies diversity in morphological form and hence of the diverse ecological adaptations that the morphologies of different embryos, larvae, and adults potentiate. This is an extremely important point: one of the most powerful approaches to discovering which functional aspects of molecular and cellular developmental processes are really fundamental, and which are peculiar to a given organism, is to make use of comparisons between organisms that develop differently. The marine environment offers many models for developmental molecular biology, and most of these have barely been superficially described. Their evolutionary relationships provide every imaginable degree of relatedness, from interspecific to interphyletic. The opportunities for startling new insights into developmental mechanisms that are awaiting exploitation in marine organisms cannot be overstressed. A series of key model marine organisms should be chosen for comprehensive molecular-level study of developmental processes throughout the life cycle. These model systems should have several key features, including evidence of “high connectivity,” that is, the relatedness of those molecular or cellular mechanisms that are uniquely accessible in the model system, to those basic mechanisms that are essential to progress in human medicine, or agriculture, mariculture, or ecological processes. In addition, requirements for the model systems should include some or all of the following, as appropriate: egg-to-egg laboratory culture must be feasible and not technically difficult; gene transfer into the egg should be possible; embryonic and other stages of material should be available in quantity; the material should be accessible for molecular biology, that is, it should be easy to prepare nucleic acids, nuclear extracts, cell types, mRNA; and clusters of evolutionarily related but morphologically distinct species should be available. At present, sea urchins and some teleost fish are the best known, but many additional invertebrate marine models need to be developed, including marine annelids, mollusks, other echinoderms, coelenterates, crustacea, and others. Marine organisms present ultimate versions of earlier evolutionary pathways, and a concerted effort to study the regulatory molecular biology of a set of diverse marine creatures will yield a depth of understanding of both evolutionary and developmental processes that far exceeds our present knowledge. This will undoubtedly affect, in turn, the state of knowledge in many of the most essential areas of bioscience and biotechnology. To put the matter in the broadest light, we can regard the genomes of the organisms on this earth as the most valuable informational natural resource that exists. This reservoir of biological information has been growing for about 2 billion years. A very large part of this store of information is resident in marine creatures, about which our knowledge has so far lagged. The benefits that will derive from

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MOLECULAR BIOLOGY IN MARINE SCIENCE: SCIENTIFIC QUESTIONS, TECHNOLOGICAL APPROACHES, AND PRACTICAL IMPLICATIONS deciphering and then applying this information are largely unforeseeable, just as not one could have predicted the fundamental transformations of society that followed from the discoveries of electricity or oil or calculus or the subatomic structure of the nucleus. Techniques to Address the Scientific Questions Molecular Biological Technologies: The key technologies are those required for core molecular biological investigations, as applied to marine organisms (described in Chapter 2). There area series of special problems (and also special advantages) attendant on transfer of standard molecular biological methodologies to marine systems. Once the molecules are accessible, the techniques, methods, and instrumentation, are all in hand. These technologies are based fundamentally on recombinant DNA procedures for isolating, cloning, characterizing, and expressing genes and their products; on application of nucleic acid and protein physical chemistry; on the availability of a series of medium-level expensive instruments, including protein microsequencers, fluorimetry, spectrophotometers, DNA sequencers, PCR machines, and phosphorimaging devices; and on optical equipment capable of image processing, such as videomicroscope image processors equipped with fluorescent and differential interference contrast optics. Preparative Methods: Development of marine molecular biology will, in the near future, require inventions of preparative methods, that is, knowledge of how to get from the organism or its cells to the molecules, genes, and proteins that must be studied. Among the preparations required are cell nuclei, nuclear extracts (that contain gene regulatory factors), mRNA, specific cell types, and cytoplasmic factors. Culture Methods: A major area of technology for marine organisms that will be required is laboratory culture devices and procedures that will permit egg-to-egg culture of various marine species, including methods for raising embryos, larvae, juveniles, and adults in large quantities. This will also potentiate genetic techniques, including development of inbred strains and isolation and generation of useful and informative mutants; control of sexual differentiation and reproduction; and among the most important objectives, exploration of physiological responses to environmental factors of every kind, chemical, physical, and biological. Laboratory culture methods are also required for study of marine pathobiology and the immune systems of marine organisms.

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MOLECULAR BIOLOGY IN MARINE SCIENCE: SCIENTIFIC QUESTIONS, TECHNOLOGICAL APPROACHES, AND PRACTICAL IMPLICATIONS Finally, there are several key technologies that need to be worked out for almost any sophisticated molecular biological application. These are development of methods for gene transfer and development of methods for tissue culture. Marine cell lines are yet very sparse, and even short-term culture methods are rare. However, for both biotechnological and basic research purposes this is an essential area, because cultured cells provide enormous opportunities for experimental manipulation, expression cloning, cell genetics, and tests of molecular function of all kinds. Gene transfer systems that are easy and sufficient exist so far only for some teleost fish and for sea urchin eggs. This technology is obviously required for analysis of gene regulatory systems, the key, basic area of research that will ultimately yield the ability to both understand and control gene expression. Practical Implications Human Health Marine biologists, employing marine organisms as model systems, have not only changed our fundamental understanding of the basic biology of marine organisms but have also provided insight concerning the basic biology of humans, including the molecular mechanisms that regulate growth, reproduction, development, and the synthesis of a host of biomedically valuable metabolites. This research has resulted in many discoveries with significance for human health and medical research. Continued and future research in this field will almost certainly lead to opportunities in the development of new tools for diagnosing, treating, and preventing disease. Marine Biotechnology Numerous opportunities can be foreseen in the area of marine biotechnology. Marine biotechnology has scarcely begun to be exploited. Marine organisms present an enormous range of interesting and useful new materials, many of which are the products of a number of genes working together during the development of the animals, for example, protein-mineral complexes with fascinating tensile properties; medically active components, signal transduction devices, regulatory devices, optical devices, phosphorescent and luminescent systems, chemical and biochemical processes, prosthetic devices, underwater films, and fabrics. When scientists have a more complete understanding of the biology of marine organisms, the gene complexes and cell types that make these substances and biosystem elements will become accessible for controlled manufacture. These applications have the potential of effecting enormous transformations in the diversity and practical usefulness of commercial biotechnology.

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