last update - January 29, 2008 MDIBL Research Mentors are research faculty from small colleges, large universities, research institutions and medical schools from around the United States and several foreign countries. Several mentors are year-round investigators at MDIBL, most are in residence only during the summer months. Listed below are the mentors and their research projects. Projects are also listed by broad research area, including neurobiology, developmental biology, marine physiology, etc. When applying for a research fellowship, students are asked to select a mentor from the list below. When a final fellowship is awarded, the Office of Education is responsible for final student-mentor assignments. Mentors not only work with students in the laboratory, but often meet in informal settings like the MDIBL Dining Hall.

REGULATION OF LIFE-LONG NEUROGENESIS
Barbara S. Beltz, Ph.D., Susan M. Hallowell and Ruby Frances Howe Farwell Professor in Biology, Wellesley College.

New neurons are born throughout life in the human brain, as well as in the brains of many vertebrate and invertebrate organisms. Work in the Beltz lab explores this process at several levels: (1) defining factors that influence the rate of neuronal birth, (2) analyzing specific effects of serotonin on the lineage of cells producing new neurons, (3) examining the differentiation of the newborn neurons. The crustacean brain is the focus of study because neuronal proliferation persists througout life in the brain of these animals and is regulated by serotonin, nitric oxide, and the day/night cycle. Furthermore, the brains of lobsters and crabs are amenable to electrophysiological analyses, and our ultimate goal is to define the neural pathway(s) that are involved in the regulation of neuronal proliferation in these organisms. Because the factors that influence neuogenesis appear to be evolutionarily conserved, these studies may illuminate mechanisms that are relevant to the variety of species that undergo life-long neurogenesis. Our most recent work at MDIBL has focused on the influences of omega-3 fatty acids and of organophosphate pesticides on the birth of new neurons in adult organisms. Undergraduates are involved in every aspect of our work, from BrdU and immunocytochemical labeling, to assessing the variety of factors influencing the rate of neurogenesis and the analysis and presentation of data. Physiology, neurobiology, developmental biology, toxicology.

CELLULAR DEFENSES AGAINST TOXIC CHEMICALS
Ned Ballatori, Ph.D., Professor of Toxicology, University of Rochester School of Medicine.

Cellular detoxification of foreign chemicals (xenobiotics) and reactive metabolic intermediates is usually accomplished by enzyme-mediated biotransformation followed by extrusion of these chemicals from the cell by specific membrane carrier proteins. The major goal of our laboratory is to identify and characterize membrane transport proteins that mediate extrusion of xenobiotics from the cell. We are studying two broad classes of xenobiotics: heavy metals, and electrophilic compounds that are conjugated with the endogenous tripeptide glutathione. Our studies are focused on liver parenchymal cells or hepatocytes, which function as the major site for the metabolism and elimination of a variety of drugs and xenobiotics. For our studies we utilize a unique non-mammalian liver cell culture model, hepatocytes isolated from Leucoraja erinacea, the little skate. Toxicology, physiology

DEVELOPING MARINE CELL LINES AND STEM CELLS
David W. Barnes, Ph.D., Senior Scientist, MDI Biological Laboratory; Director, Marine Cell Lines and Stem Cell Program

The laboratory is involved in the development of indefinitely proliferating fish and marine invertebrate cell lines. Our approach is to limit the amount of serum added to the culture medium and to supplements with peptide growth factors and other biological activities. We have developed systems for culture of zebrafish embryonic and adult tissues and have used these in developmental and toxicological studies. We also have applied this approach to marine models including pufferfish (fugu), sharks and skates. Cell culture, stem cells

MECHANISMS OF XENOBIOTIC TRANSPORT AND EXCRETION IN THE LIVER
James L. Boyer, M.D., Professor of Medicine; Director, Liver Center; Chief, Division of Digestive Diseases; Yale University School of Medicine

The liver is an organ with many functions, one of which is to defend the body against the accumulation of xenobiotics and environmental toxicants. My laboratory is interested in how the liver functions as an organ of excretion; specifically how xenobiotics and environmental toxicants are taken up by the liver, metabolized, and eliminated into bile. In addition, we are interested in how the transport systems and enzymes that provide these functions undergo adaptive regulation when exposed to toxic compounds or when the bile secretory process is impaired in diseases that produce cholestasis. The later proccess involves transcriptional regulation and the role of nuclear receptors. At MDIBL, together with my colleague Ned Ballatori, we utilize the marine skate as an experimental model to study this process. This comparative toxicogenomics approach is designed to provide insight into the critical determinants of these defense mechanisms in human liver. Toxicology, physiology

BEHAVIORAL CONTROL IN DECAPOD CRUSTACEANS
Andrew E. Christie, Ph.D., Research Scientist and Lecturer, Department of Biology, University of Washington

(1) Elucidation of the molecular and cellular components of crustacean circadian-circatidal clocks. Virtually all species express circadian rhythms. The persistence of these rhythms under constant laboratory conditions is taken as evidence for the existence of an innate, self-sustaining timekeeping mechanism, i.e. a biological clock. Furthermore, species from intertidal habitats often express circatidal rhythms; oscillations in behavior and physiology attuned to the tidal cycle and which also rely on a biological clock. Given that circadian rhythms are present in almost all extant organisms, including prokaryotes, the presence of two biological timing systems oscillating with different periods in intertidal organisms raises the interesting possibility that the circatidal system may have evolved from a pre-existing circadian system and might utilize the same molecular and cellular machinery. As a first step towards testing this hypothesis we have begun to identify the molecular components and the neural substrates for circadian and circatidal oscillators in decapod crustaceans. Behavioral circadian and circatidal rhythms have been studied in several intertidal invertebrates, including mollusks, annelids and crustaceans, but little is known about the neural and molecular components of these biological timing systems. Of these animal taxa, the neurochemistry and neuroanatomy of decapod crustaceans has been particularly well established and therefore these animals represent an ideal model in which to search for these components. Moreover, the close phylogenetic relationship between crustaceans and insects (where both molecular and cellular components of the circadian system are well established in a number of species; e.g. Drosophila) provides us with background information and biological reagents to undertake this study. We believe our experiments will not only contribute to the understanding of the molecular and neural pathways by which intertidal organisms are able to synchronize their physiology and behavior to environmental cycles of different periods, but will also help to determine whether common threads exist between biological timing systems that emerged in response to different environmental needs.

(2) Identification and characterization of novel peptide hormones.  My research team has begun a long-term study to identify novel peptide hormones in decapod crustaceans using a combination of molecular cloning and biological mass spectrometry.  Our ultimate goal is to provide a better understanding of the complement of peptide used as endocrine/paracrine agents in this commercially important group of animals, as well as to provide insight into the evolution of peptidergic signaling systems in general.  For our peptide surveys, we are using both neuroendocrine and midgut tissues, the latter included as we have recently identified the crustacean midgut as a major endocrine organ in decapods. Behavior, comparative genomics, evolutionary biology, environmental biology, marine physiology, neurobiology

THE MOLECULAR AND BIOCHEMICAL PHYSIOLOGY OF H+ EXCRETION IN THE GILLS OF MARINE FISH
James B. Claiborne, Ph.D., Professor of Biology, Georgia Southern University.

The fish gill is a multi-purpose organ, carrying out respiration, the elimination of nitrogenous wastes, osmoregulation and the regulation of acid (H+) within the blood of the animal. Little is known about the actual cellular mechanisms of acid transfer which take place within the gill tissue. We have hypothesized that saltwater fish may excrete acid using a family of sodium hydrogen exchanger proteins (NHE) within the gills. In this project we utilize immunological and molecular analyses to study these gill mechanisms in several species of marine fish. Our preliminary findings suggest that fish may possess at least three different members of the NHE family homologous to the mammalian types, but unlike the mammalian version, one of these isoforms (NHE-1) may play an important role in systemic recovery from acidosis. A second isoform (NHE-2) has only previously been found in mammalian species and is thought to be important in intestinal function. We now have the first evidence that this protein is also synthesized in the fish gill, and have recently cloned the full length, open reading frame, of the cDNA for this transporter. A determination of NHE function within these animals will not only provide new insight into the physiology of gill acid-base and ion regulation, but also may shed light on evolutionary relationships between the vertebrates. Further, since we know that the cellular mechanisms within the gills are similar to those currently being studied in the mammalian kidney, new insight into the physiology of the kidney and other organs may also be elucidated. Marine physiology

PHYSIOGENOMIC CONTROL OF SEA URCHIN EMBRYOGENESIS
James A. Coffman, Ph.D., Investigator, MDIBL.

We are interested in how cell fate is specified during animal development, and how this process is controlled by the interactions between genetic information, cell signaling, physiology, and the environment.  We use sea urchin embryos as a model system to study this problem, because they are accessible to experimental manipulation at many different levels, and their genome has been sequenced and annotated.  Currently we have two specific projects ongoing in the lab.  One project seeks to determine the molecular and cellular mechanisms by which Runx proteins, a highly-conserved family of transcription factors, control the proliferation and survival of cells during development.  One of the long term goals of this project is to understand why Runx genes are often associated with human cancers such as leukemia, wherein cells proliferate out of control and do not respond to cell-death signals.  This project uses specific-knockdown of a sea urchin Runx protein to facilitate determination of what processes and genes are normally under its control.  The second project aims to understand the symmetry-breaking process that establishes the oral-aboral (anterior-posterior) axis of the sea urchin larva.  We are testing the hypothesis that an asymmetric distribution of mitochondria in the sea urchin egg specifies this axis through redox signals (e.g., reactive oxygen species) that modulate the activities of regulatory proteins such as transcription factors, kinases, and/or phosphatases.  We are currently developing a number of pharmacological and molecular reagents for testing this hypothesis.  Both projects provide opportunities for student research involving microscopy and imaging, microinjection, and molecular techniques such as polymerase chain reaction (PCR). Developmental Biology

MACHINE LEARNING AND BIOINFORMATICS
Clare Bates Congdon, Ph.D., Assistant Professor, Computer Science Department, Colby College

Machine learning is a subdiscipline of computer science that addresses how to extract patterns from data in order to make inferences or predictions. Our work focuses on the use of genetic algorithms, in which one "evolves" the model used to represent the patterns. In collaboration with Dr. Carolyn Mattingly and Dr. H. Rex Gaskins, we have developed GAMI, a genetic algorithms approach to motif inference. Our work focuses on the search for putative conserved regulatory regions in non-coding DNA. In addition to GAMI, we have also developed Gaphyl, a genetic algorithms approach to inferring phylogenies. We also work on developing genetic algorithms approaches to data mining. Undergraduate students have contributed to all of these projects. Bioinformatics

MOLECULAR COMPARISONS OF CORNEAL AND CONJUNCTIVAL SUTURAL FIBER FORMATION MECHANISMS IN AVIAN AND ELASMOBRANCH EMBRYOS
Gary W. Conrad, Ph.D., and A.H. Conrad, Ph.D., Division of Biology, Kansas State University

LASIK surgery is an increasingly popular vision-corrective procedure in which an epithelial flap is lifted from the cornea surface, underlying stroma matrix and cells are removed by laser, and the epithelial flap is laid back over the stroma. Recent work from our lab and that of Dr. Henry Edelhauser [former MDIBL summer scientist] has shown that this flap never properly fuses with the underlying stroma. The LASIK patient remains permanently at risk that any sudden head jar will dislodge the flap lift, causing immediate visual dysfunction. Our previous work has shown that corneas in organisms that form no corneal endothelium, such as the skate Raja eglanteria, grow sutural fibers that extend from the basement membrane underlying the corneal epithelium perpendicularly down into the corneal stroma. These sutural fibers stabilize corneal thickness through very large osmotic changes. Similar sutural fibers have been observed in chick cornea epithelia, scleral papillae, and underneath feather papillae in chick skin. Intriguingly, Sonic Hedgehog-induced SWiP-1 is expressed in both chick scleral papillae and embryonic feather as they are forming their sutural-like fibers. The molecular steps by which chick and skate corneas induce sutural fibers to form, and chick sclera papillae and skin feather germs induce sutural-like fibers to form, if initiated in LASIK epithelial flaps, may induce sutural fibers to grow down from LASIK flap epithelial basement membranes to permanently secure the LASIK flap tissue to the underlying corneal stroma following LASIK surgery. Our hypotheses are: 1) Molecular steps by which embryonic skate corneal epithelium forms many sutural fibers in the underlying corneal stroma and perpendicular to it - will be the same steps as used by embryonic avian conjunctival epithelial papillae AND feather germ placodes to form collagen fibrils perpendicular to the surface; 2) Embryonic skate corneal epithelium, if applied to the de-epithelialized stromal surface of embryonic chick cornea or dermis, will send sutural fibers down into the extracellular matrix (ECM) of those stromal preparations. Developmental biology, marine physiology

EFFECTS OF ACCLIMATION TEMPERATURES ON MEMBRANE SUSCEPTIBILITY TO LIPID PEROXIDATION: BIOPHYSICAL AND FUNCTIONAL CONSEQUENCES
Elizabeth L. Crockett, Ph.D., Department of Biological Sciences, Ohio University

A consequence of life in the presence of oxygen is the regular production of free radicals and other reactive oxygen species (ROS). Some ROS are of sufficient energy to initiate lipid peroxidation (LPO), a self-propagating process with the potential to damage biological membranes. Elevated levels of phosphatidylethanolamine (a common phospholipid) and polyunsaturated fatty acids in membranes from animals living at low body temperatures raise the likelihood that susceptibility to LPO (when measured at a common temperature) may be greater than in membranes from animals living at warm temperatures. Greater susceptibility to LPO at low temperatures may, however, be offset by reduced rates of ROS production and LPO in the cold. These scenarios raise the possibility that membrane susceptibility to LPO is conserved at physiological body temperatures. While LPO is intensively studied in biomedicine and aging biology, its significance in the contexts of temperature and adaptational physiology has yet to be elucidated. The research objectives in Dr. Crockett’s lab include 1) assessment of whether membrane susceptibility to LPO is maintained at physiological temperatures, 2) quantification of LPO products, oxidant activity, and antioxidant defense, and 3) analyses of the effects of LPO on membrane biophysical (e.g., membrane fluidity) and functional (e.g., activities of membrane transport proteins) properties in thermally-acclimated animals. marine physiology

PRIMITIVE EXTANT FISHES: THE KEY TO THE EVOLUTION OF Na+/H+ EXCHANGER GENE FAMILY?
Susan Edwards, Ph.D., Associate Professor, Department of Physiology and Pharmacology, James Cook University

The ability of an organism to regulate cellular pH, volume and ion composition is critical to its survival. One such mechanism is the transmembrane exchange of sodium ions for proton ions. This exchange is seen in all phyla and kingdoms and is responsible for the strict homeostatic control of these two ions. The sodium/proton exchanger (NHE) gene family plays a role in a diverse range of physiological processes and in mammals NHE dysfunction is related to a number of pathophysiological processes. The primary purpose of my research is to explore the evolutionary relationships that may exist between members of the NHE plasma membrane sub-group responsible for acid/base regulation in lower vertebrates such as the Agnathan fishes and higher vertbrates. The comparative biology methods used include molecular biology, protein biochemistry, and in vivo physiology to examine the role of NHE genes in the lower vertebrates, the fishes.

MECHANISM AND CONTROL OF SECRETION OF CHLORIDE BY THE RECTAL GLAND OF THE SHARK
Franklin H. Epstein, M.D., William Applebaum Professor of Medicine, Harvard Medical School and Patricio Silva, M.D., Associate Professor of Medicine, Harvard Medical School; Chief, Division of Nephrology, New England Deaconess Hospital and Joslin Diabetes Center

Atrial natriuretic peptide (ANP) is the principal hormone regulating the salt content of the shark through its control of active secretion of sodium chloride by the rectal gland. We have shown that it exerts its effect through the release of vasoactive intestinal peptide (VIP) from nerve cells within the gland and it is VIP that stimulates the cells to secrete chloride. The mechanisms that regulate the secretion of ANP from the shark heart are currently being studied. The rectal gland nerves contain many neurotransmitters in addition to VIP. These include somatistatin, bombesin, cholecystokinin, galanin, catecholamines among others. The role of only some of these peptides on the secretion of chloride by the rectal gland has been studied. Many of these peptides have an inhibitory effect. The effect of these peptides is currently being studied. The preparations used in the laboratory are: isolated perfused shark hearts; isolated perfused rectal glands; rectal gland cross perfusions; isolated rectal gland tubules; cultured rectal gland cells; subcellular fractionation of rectal glands. These preparations are used for transport studies, enzyme measurements, oxygen consumption, binding assays, and radioimmunoassays. Marine physiology

PARACRINE CONTROL OF FISH GILL FUNCTION
David H. Evans, Ph.D., Professor of Zoology, University of Florida.

Since the diameter of blood vessels can control blood pressure and flow, there is considerable interest in the role of various substances in controlling the contraction vs. dilation of blood vessels in vertebrates, especially humans and other mammals. In fishes, the control of blood flow to the gills can affect important functions of the gills including gas exchange, osmoregulation, nitrogen excretion, and acid-base balance. Our interests center around the role of various substances (endothelin, nitric oxide, prostaglandins, and natriuretic peptides) that may affect the perfusion of the gills as well as active salt secretion. Our studies use physiological, pharamacological, and some molecular techniques. Marine physiology

CLONING, EXPRESSION, AND REGULATION OF RECEPTORS AND CHANNELS INVOLVED IN CHLORIDE TRANSPORT IN THE SHARK RECTAL GLAND
John N. Forrest, Jr., M.D., Director, MDI Biological Laboratory; Professor of Medicine, Nephrology Division, and Director, Office of Student Research, Yale University School of Medicine.

The rectal gland of the primitive dogfish shark is an ideal model system for it contains a high density of the receptors and channels that regulate chloride secretion in higher vertebrates, including man. Our laboratory has used both physiological and molecular biological approaches to investigate peptides, G-protein coupled receptors and channels involved in chloride transport in this tissue. We cloned the first gene identified in the shark and found that this gene encodes for a new class of heart natriuretic peptides (CNP) not previously found in cardiac tissue. We have also recently cloned from the rectal gland: (1) the membrane receptor for CNP; (2) a unique adenosine receptor that regulates chloride secretion; and (3) the full length gene for shark CFTR, the gene that is defective in cystic fibrosis. Using integrated methods that include perfusion studies, electrophysiological measurements, PCR, oocyte expression studies, and immunohistochemistry, our goal is to understand the interaction of these proteins in tissues of higher species, including the mammalian kidney and lung. We are also studying the effects of environmental toxins on signal transduction in the rectal gland and the role of tyrosine phosphorylation in the regulation of CFTR. Techniques include: preparation of RNA, DNA, PCR, gene cloning and sequencing, receptor binding studies, perfusion of the isolated shark rectal gland, cell culture, oocyte expression studies, immunohistochemistry, Western blots, and confocal microscopy. Functional genomics, toxicology

TOXICOGENOMIC ANALYSIS OF CIONA INTESTINALIS AND ITS INTESTINAL MICROBIOTA
H. Rex Gaskins, Ph.D., Professor of Immunobiology, Unversity of Illinois at Urbana-Champaign

We are studying the ascidican tunicate Ciona intestinalis and its intestinal microbiota. The critical evolutionary position of ascidians as basal chordates and the simplicity of their embryogenesis have attracted developmental and evolutionary biologists since the turn of the 20th century. Because of this, the genomes of C. intestinalis as well as its close relative C. savignyi were fully sequenced recently. These animals are filter feeders and live attached to submerged substrates in marine waters where they encounter high concentrations of complex polyphenols, halogenated aromatics, methylated sulfides, and some heavy metals. Little is known about the mechanisms by which tunicates detoxify or otherwise tolerate these natural toxins in the marine environment. We are addressing the hypothesis that filter-feeding tunicates such as Ciona rely on bacterial symbionts associated with the branchial sac for this purpose. The branchial sac is an enlarged pharnyx whose wall is perforated by numerous tiny gill slits that serves as both a filter feeding device and a respiratory organ. The research involves characterization of the microbes that are stably associated with the branchial sac as well as identification of the detoxification gene families harbored by these microbes. A comparative genomic approach is used to pursue our long-term goal of determining whether the support by C. intestinalis of a bacterial metagenome that contributes to detoxification has influenced, over evolutionary time, its complement of detoxification genes. Bioinformatics, Environmental Biology, Functional Genomics, Toxicology/Toxicogenomics

CELL VOLUME REGULATION IN SKATE RBC
Leon Goldstein, Ph.D., Vice-Chair, Department of Molecular Pharmacology, Physiology and Biotechnology, Brown University.

We are using a combination of molecular biological and cell physiological techniques to investigate how cells maintain their volume constant when challenged by osmotic stress. Our studies are focused on the mechanisms of cell membrane transport and signal transduction that are involved in the cell’s response to hypotonic stress. The erythrocyte of the osmotically tolerant skate provides an excellent model system in which to study these questions. Marine physiology

IDENTIFICATION OF GENES EXPRESSED IN THE DEVELOPING RENAL TISSUE OF THE SPINY DOGFISH, SQUALUS ACANTHIAS
Hermann Haller, M.D., Director of the Dept. of Nephrology, Clinic for Internal Medicine, Hannover Medical School, Germany

Nephroneogenesis, or the growth of new nephrons (the functional unit of the kidney), does not terminate with birth in skates and sharks. Mature and developing nephrons lie side by side in sexually mature animals. Our current project involves the isolation of genes responsible for the development of new nephrons in the kidneys of sharks and skates. After tissue from the developing zones in the kidney is extracted the RNA is isolated, cDNA is cloned and printed onto micro array filters. Developing genes that show differential displacement to mature genes are sequenced and BLAST searched. The genes of interest are then checked with real-time PCR and those genes that show a difference in expression are further explored with insitu hybridization. Another aspect we are studying is the stimulation of growth in the kidney tissue of live skates after a partial nephrectomy. The tissue from the nephrectomized animals is then prepared for immunohistochemistry. We are also investigating the continued growth of kidney tissue slices in culture media for an extended period of time. This is a year-round ongoing research project and any of the above techniques for this identification could be in progress. Marine genomics, developmental biology

STEROL LIMITATION OF ZOOPLANKTON GROWTH: ROLES IN NUTRITION AND MEMBRANE BIOLOGY
R.P. Hassett, Ph.D., Department of Biological Sciences, Ohio University

To understand nutritional limitations for zooplankton growth it is necessary to identify key components in the diet and define the conditions under which these components become limiting. Cholesterol is the most abundant neutral lipid in the plasma membranes of animals, and is therefore a requisite for building new membrane. Sterols are prime candidates for limiting zooplankton growth, not only because of cholesterol’s fundamental roles in plasma membranes, but also because the composition and concentration of sterols in phytoplankton vary tremendously. The degree to which cholesterol and other sterols limit zooplankton growth are being determined and the physiological bases for this essentiality explored. Dr. Crockett’s research encompasses a range of levels of biological organization (from ecosystem dynamics to biochemical processes), and therefore it is particularly well-suited for undergraduate student participation. Students are gaining conceptual and specific knowledge of zooplankton and temperature biology by testing hypotheses designed to elucidate the physical and biotic conditions under which cholesterol and other sterols limit zooplankton growth while at the same time clarifying the physiological underpinnings for this requirement. Marine physiology

MUSCLE DEVELOPMENT AND MORPHOGENESIS IN ZEBRAFISH
Clarissa Henry, Ph.D., Asst. Professor of Biological Sciences, The University of Maine

A large variety of diseases, both inherited and acquired, affect muscle tissues in humans. In order to prevent and/or treat such disease, it is necessary to understand the pathology at the cellular and molecular level. Because each step of muscle specification and differentiation translates to a progressive refinement of functional physiology, studying muscle development may lead to therapeutic insights. THE GOAL OF OUR LABORATORY IS TO ELUCIDATE THE SIGNALING NETWORKS THAT UNDERLIE MUSCLE MORPHOGENESIS. We study skeletal muscle morphogenesis during zebrafish development. Skeletal muscle is comprised of segmentally reiterated myotomes. Like the mammalian tendon, the zebrafish myotome boundary transduces force from muscle to the skeletal system. Thus, myotome boundary formation, as well as skeletal muscle morphogenesis, is critical for normal development and muscle function. Our research investigates the morphogenetic signaling networks that underlie skeletal muscle and myotome boundary formation. The zebrafish is an excellent model system with which to integrate the genetic, molecular, and cell biological mechanisms that underlie muscle development. Because the signaling networks that regulate muscle development are remarkably conserved among vertebrates, our studies may lead to novel insights into development of therapeutics for muscle and tendon diseases.

Cell and molecular mechanisms that underlie fast muscle cell elongation
Muscle precursor cells begin as rounded cells that elongate to form long muscle fibers. This elongation is necessary for efficient actin-mediated contractility and thus normal muscle function. However, it is not known either how muscle precursor cells elongate or what proteins are required for muscle cell elongation. We are studying this important question.

Cell and molecular mechanisms that underlie myotome boundary morphogenesis
The myotome boundary functions as the major attachment site for skeletal muscle fibers and transmits force to the skeletal system. Despite its critical importance, myotome boundary morphogenesis is not well understood. We are currently investigating signaling pathways involved in myotome boundary morphogenesis.

ADAPTATION OF MARINE INVERTEBRATES TO EXTREME ENVIRONMENTS: ESTUARINE AND INTERTIDAL CONDITIONS
Raymond P. Henry, Ph.D., Professor, Department of Biological Sciences, Auburn University.

Response of carbonic anhydrase gene expression to low salinity stress: induction and regulation. The current focus of Dr. Henry's laboratory is on the mechanism of regulation of carbonic anhydrase (CA) gene expression in gills of euryhaline crustaceans in response to low environmental salinity (i.e., when crustaceans migrate from the open ocean into estuaries as part of their annual life cycle). Protein-specific CA activity is induced 8-14 fold, depending on species and degree of low salinity exposure, and this induction has been shown to occur at the transcriptional level. CA mRNA increases six fold by 24 hr after transfer to low salinity, and CA activity increases between 48 and 96 hr post-transfer. The regulation of CA expression and induction appears to be under inhibitory control via a putative CA gene repressor found in the major endocrine complex of the crab, the eyestalk. A combination of approaches and techniques, from classical endocrinology to molecular biology, is used to identify the CA repressor and understand its functions. Because this project can be approached at different levels, many aspects of the research are ideal for undergraduate participation. The basic concepts of endocrinology and new hormone identification are easy to grasp, and the experiments are technically straightforward. Students become familiar with the process of how new hormones are identified by use of classical examples from the scientific literature (e.g., insect juvenile hormone, crustacean molt inhibiting hormone) and then apply these principles and techniques to the study of the CA repressor. The students identify a specific question about the CA repressor (e.g., is it a peptide or a steroid?) and then formulate a series of experiments, based on known concepts and techniques, to answer that question. Also, since these types of questions are typically very specific, they have a good chance of being answered in the amount of time that a summer student would have available.

Respiratory and acid-base adaptations to emersion during low tide
Green crabs forage in clam and mussel beds that get exposed to air during low tides. The crabs remain in the intertidal zone, under rocks and seaweed, but experience up to 6 hours of air exposure. The focus of this project is on the adaptations that allow the crab to maintain adequate gas exchange (O2 uptake and CO2 excretion), hemolymph pH regulation, and waste excretion while they are cut off from their normal aquatic habitat. Marine genomics, environmental biology, marine physiology

THE STURCTURE AND FUNCTION OF CYTOSKELETAL PROTEINS IN SEA URCHINS AND ELASMOBRANCHS
John Henson, Ph.D., Associate Professor of Biology, Dickinson College.

Dr. Henson's research program is highly accessible to undergraduates as is evidenced by the large number of undergraduate coauthors appearing on his publications. Dr. Henson studies the mechanisms underlying the process of actin-based retrograde flow in sea urchin coelomocytes. Retrograde flow has been indicated as important in cellular translocation, in the targeting of cellular migrations, in cell-substrate interactions, and in modulating crosstalk between actin and microtubules; however, the exact mechanisms and regulation of this fundamental process are largely unknown. Current research objectives include the characterization of the roles of actin binding and motor proteins as well as an effort to study the inducible transformation of these cells from a lamellipodial to a filopodial form. Opportunities for undergraduate independent research include projects involving sophisticated microscopic imaging and biochemical and immunological-based methods. Examples of current undergraduate research efforts include the manipulation of actin cytoskeletal organization using a combination of the drug BDM and osmotic shock and an attempt to determine the phagocytic potential of sea urchin coelomocytes. Cellular biology, Marine physiology

EXPRESSION AND FUNCTIONAL CHARACTERIZATION OF AQUAPORINS FROM THE EEL, Anguilla anguilla
Warren G. Hill, Ph.D., Research Assistant Professor, University of Pittsburgh School of Medicine

Osmoregulation is the physiological process by which animals regulate their internal fluid environment and maintain blood and cellular fluid compositions within extremely tight limits, despite the presencce of a changing environment. Aquaporin proteins play a crucial roel in osmoregulation. Aquaporins (AQPs), the study of which was awarded the 2003 Nobel Prize in Chemistry, are a family of water channels embedded within the membrane of epithelial cells which allow highly specific transport of water molecules. Some AQPs transport other substrates like glycerol and urea and possibly gases like carbon dioxide. We are studying the biophysical transport properties of a series of AQPs cloned from the American eel. The eel is a euryhaline teleost fish which means it employs adaptive mechanisms which allow it to survive in fresh or salt water. It has been shown that AQPs present in the gut are upregulated upon salt water adaptation but downregulated in the gill. We wish to characterize the transport specificities and single channel conductances of several of the more interesting aquaporins from the eel. We use the Xenopus (African clawed frog) oocyte expression system to explore these parameters. Students will microinject RNA coding for specific AQPs into oocytes and then study water and substrate transport kinetics by video microscopy, subcellular fractionation, isolation of the plasma membrane and stopped-flow flurometry. They will also actively participate in data analysis and experimental design. Marine physiology

OSMOREGULATION IN FUNDULUS HETEROCLITUS
George W. Kidder III, Ph.D., Senior Investigator, Mount Desert Island Biological Laboratory

When estuarine Killifish live in fresh water, they absorb water by osmosis, and must use metabolic energy to return this water to the stream. The same fish in sea water loose water by osmosis, and must "pump" water into its body. When the fish are in water with the same osmotic concentration as its body fluids, they are freed from this expenditure of energy, which should give it a selective advantage. Studies using a combination of laboratory and field techniques are underway to determine whether Killifish prefer iso-osmotic water, to measure the amount of metabolic energy actually required for water movement under various conditions, and to identify and quantitate the changes in transport proteins which are responsible for the change from fresh water to salt water acclimation. Marine physiology, behavior, ecology

SENSORY RECEPTION AND PREDATOR EVASION IN CRUSTACEAN ZOOPLANKTON
Petra H. Lenz, Ph.D., Associate Researcher, Pacific Biomedical Research Center, University of Hawaii, Manoa.

Predation is often the greatest source of mortality for planktonic organisms, and marine organisms have met this challenge in many ways. Calanoid copepods (Crustacea) have an escape performance that is matched by few other organisms. Underlying this performance is an array of unusual neuromotor characteristics evolved in response to predation pressure, including high mechanoreceptive sensitivity, high neuronal firing-frequency capabilities and the occurrence of myelinated nervous systems in about half of all calanoids. Behaviorally it includes fast reactions to mechanical stimuli, high output of muscle energy and high cycle rates of muscle action. Conventional crustacean physiological properties cannot account for copepod escape capabilities. Dr. Lenz investigates how these animals achieve their remarkable behavioral and physiological performance using an integrated approach. Because this project can be divided into smaller components, it has been ideally suited for the involvement of undergraduate students doing research projects. In the past, students have quantified escape responses to photic and hydrodynamic stimuli. Morphological studies have involved both electron microscopy, and light and fluorescence microscopy. Most recently, students have investigated the stress response using molecular techniques. Weekly meetings allow students to present their progress to their peers and understand how their work fits into the overall laboratory research goals. Marine physiology, behavior, marine genomics

BIOINFORMATICS: THE COMPARATIVE TOXICOGENOMICS DATABASE
Carolyn Mattingly, Ph.D., Director, Comparative Toxicogenomics Database, MDIBL

According to the Environmental Protection Agency, there are more than 75,000 chemicals used in commerce today; however, the toxic potential of these chemicals and the mechanisms by which they affect living organisms are not well understood. Many chemicals are toxic because they disrupt the normal expression of genes or protein functions. To promote understanding about which genes and proteins are targeted by environmental chemicals, the Mount Desert Island Biological Laboratory is developing the Comparative Toxicogenomics Database (CTD). CTD will be community-supported, and publicly available through a WWW interface. It will be the first resource to: 1) describe associations between specific genes, proteins and toxic agents; 2) provide gene and protein sequences from diverse species with a focus on aquatic and mammalian organisms; and 3) offer a range of computational tools for comparative sequence analysis. These features will provide a collective perspective of existing data and facilitate cross-species comparisons of genes and proteins that are affected by toxic agents. The goal is to promote understanding about molecular mechanisms of toxicity, explain why some chemicals are only toxic in certain species, and provide insight about the complex interactions between the environment and human health. Bioinformatics, toxicology

THE CELLULAR AND MOLECULAR BIOLOGY OF XENOBIOTIC TRANSPORT
David S. Miller, Ph.D., Senior Investigator, Laboratory of Pharmacology and Chemistry, NIH, National Institute of Environmental Health Sciences

Excretory and barrier tissues, e.g., kidney, liver, blood-brain barrier, express potent xenobiotic efflux pumps that are primary determinants of xenobiotic uptake, distribution and excretion. As such they influence both the efficacy of therapeutic drugs and the toxicity of pollutants and pollutant metabolites. We use comparative models, fluorescent compounds and confocal fluorescence microscopy to characterize the cellular mechanisms that drive xenobiotic transport in intact kidney tubules, choroid plexus and brain capillaries. The current focus is on the short-term and long-term control of ATP-driven xenobiotic pumps, e.g., p-glycoprotein and Mrp2. Physiology, toxicology

SIGNAL TRANSDUCTION AND TRANSCRIPTION REGULATORY ELEMENTS IN VERTEBRATE SPERMATOGENESIS
Antonio Planchart, Ph.D., Investigator, Mount Desert Island Biological Laboratory and College of the Atlantic.

The focus of our lab's work is to understand the molecular regulation of spermatogenesis, using the spiny dogfish shark. Spermatogenesis exhibits many interesting aspects of cell biology, including renewal of a stem cell population (spermatogonia), and cellular crosstalk between supporting cells and spermatogonia. It also involves a developmental program that produces terminally differentiated spermatozoa and it contributes to population diversity by menas of meiotic recombination. One aspect of our research assumes that clusters of spermatogenesis-specific genes are transcriptionally regulated by common approaches to identify and test potential regulatory elements involved in this process. A second aspect of our research is geared to understand the role of phosphatidylinositol polyphosphate phosphates in sperm development and function. The phosphoinositide signal transduction pathway is a classical second messenger system and it appears to be required for either sperm development or function. In our lab, undergraduates have their own project and are expected to participate in all aspects of its development. They receive training in various techniques, from cell culture to protein purification, and participate in data analysis and presentation. Cell biology, developmental biology, and stem cells

PHYSIOLOGY OF D-AMINO ACIDS IN MARINE INVERTEBRATES
Robert L. Preston, Ph.D., Professor of Physiology, Department of Biological Sciences, Illinois State University.

Many marine invertebrate tissues (40% of 140 species in 12 phyla examined) contain high concentrations of free D-amino acids in their cell cytosol. This is surprising considering that it is generally assumed that D-amino acids are "unnatural" (compared with the common L-amino acids) and are not involved in normal biological processes. It has been found, however, that D-amino acids are synthesized, metabolized and transported by marine invertebrate tissues. At least 15 species contain enzymes called racemases that interconvert D and L amino acids. Student colleagues will assist with all aspects of research including collecting and learning local marine invertebrate species from the intertidal zone and mudflats and conducting mentored independent research projects to detect and characterize D-amino acid content and associated metabolic processes. Potential projects include characterization of D-selective membrane transport systems, characterization and kinetic analysis of racemase activity in a selected species, isolation and purification of racemases, and isolation of mRNA, DNA and sequencing of racemase genes from marine invertebrate tissues. Marine physiology, molecular biology

FLOUNDER RENAL AND CHOROID PLEXUS RESPONSES TO PHYSICOCHEMICAL STRESSES
J. Larry Renfro, Ph.D., Professor of Physiology, Department of Physiology and Neurobiology, University of Connecticut.

Dr. Renfro's research is directed at understanding the transepithelial transport of inorganic and organic molecules and the messenger/pathways involved in regulating the transport processes. Ussing flux chambers and membrane vesicles are primary techniques used to examine ion transport across numerous epithelial tissues including the renal proximal tubule, intestine, and choroid plexus. Sulfate anion exchangers in the basolateral and apical membranes facilitate the movement of sulfate from interstitium to lumen. In the presence of luminal bicardonate, carbonic anhydrase (CA) activity is required to create the hydroxyl gradient necessary for basolateral sulfate/hydroxyl exchange. Dr. Renfro and his students have shown that the hormone cortisol directly increases CAII protein abundance, CA biochemical activity and CA-dependent sulfate secretion in the marine teleost proximal tubule. Undergraduate students are trained in the Ussing chamber and membrance vesicle transport techniques allowing them to develop projects that help to explore pharmacologically the relationships between anion exchangers, carbonic anhydrases, and proton pumps. Marine

SGK REGULATION OF CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR (CFTR) EXPRESSION AND FUNCTION
Denry Sato, Ph.D., Investigator, Mount Desert Island Biological Laboratory

In collaboartion with Drs. Stanton and Frizzell we are using several model systems (frog oocytes, human airway cells and zebrafish) to study the regulation of the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is the chloride ion channel protein that is defective in cystic fibrosis, which is the most common genetic disease in Caucasians. The function of CFTR is influenced by its level of synthesis, transport to the plasma membrane, activation while in the membrane, internalization, and degradation. We are studying the functional interactions between CFTR and a protein kinase called SGK, which we believe increases CFTR activity by increasing the amount of CFTR in the plasma membrane of airway epithelial cells. The project involves molecular cloning methods, biochemical assays and electrophsiological studies.

ARSENIC REGULATION OF ABC TRANSPORTER EXPRESSION AND FUNCTION
Bruce Stanton, Ph.D., Professor of Physiology, Dartmouth Medical School

The long-term objective of our research is to elucidate how arsenic, a toxic metalloid, affects xenobiotic bioavailability and increases the incidence of atherosclerotic disease, diabetes mellitus as well as several types of drug resistant cancers. Our studies will focus on elucidating the effects of low, environmentally relevant levels of arsenic on the expression and function of two ABC (ATP Binding Cassette) transporters: the cystic fibrosis transmembrane conductance regulator (CFTR) and the multidrug resistance protein 2 (MRP2). CFTR is a cAMP-activated Cl channel, and plays an important role in salt homeostasis. MRP2 transports xenobiotics, including chemotherapeutic drugs, toxins, and arsenic-glutathione conjugates out of cells and, thereby, plays a role in xenobiotic excretion in bile and urine, protects the brain from xenobiotic and toxic compounds and limits the intestinal absorption of drugs. In preliminary studies we report that environmentally relevant levels of arsenic blocks the ability of CFTR to regulate salt balance in the teleost Fundulus heteroclitus. By contrast, we found that arsenic increases MRP2 expression and function in kidney and intestine.  Studies will be conducted to elucidate the effects of arsenic on hormonal regulation of ABC transporter expression and function in two model systems: the euryhaline teleost Fundulus heteroclitus, and in polarized human colonic Caco-2 epithelial cells. The hypothesis to be tested is that arsenic modulates hormonal regulation of ABC transporter gene expression and function. Toxicology, Toxicogenomics

FUNCTIONAL GENOMICS OF ENVIRONMENTAL ADAPTATIONS IN MARINE CRUSTACEANS
David W. Towle, Ph.D., Senior Scientist and Director of the Marine DNA Sequencing Center, Mount Desert Island Biological Laboratory

The dynamic environment of coastal watere presents both challenge and opportunity to its aquatic inhabitants. Salinity, temperature, oxygen concentration, and food availability may change rapidly over time and space. Our research is directed toward understanding how crabs, lobsters, and copepods alter their gene expression patterns in response to such environmental changes. Basic tools of molecular biology are used to identify new candidate genes and monitor their expression. Bioinformatics and high throughput gene sequencing coupled with DNA microarray are recent additions to the lab's repertoire, in the last two years producing over 38,000 new seqeunces from fiver different marine species as starting material for hypothesis-driven research. Marine functional genomics