Whole Genome Sequencing of the Little Skate (Raja erinacea):
Elasmobranch Functional Genomics
David Barnes, Ph.D.
| Section 1 |
Elasmobranch genomics: providing a better connection between human and non-human organisms |
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| Section 2 |
The role of the Mount Desert Island Biological Laboratory |
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| Section 3 |
The contribution of skates and sharks to improving human health and understanding disease |
| Section 4 |
Selected recent citations: Biomedical research and skates |
| Section 5 |
Selected recent citations: Comparative genomics and elasmobranch research in toxicology and medicine |
| Section 6 |
Selected earlier citations: Shark and skate research at Mount Desert Island Biological Laboratory |
Elasmobranch genomics: providing a better connection between human and non-human organisms.
Chondrichthyes fishes appeared 450 million years ago and today are the oldest existing gnathostomes, or jawed vertebrates. The elasmobranchs (sharks, rays and skates), which comprise most chondrichthyan organisms, diverged from the line leading to actinopterygian and sarcopterygian fishes over 400 million years ago. Elasmobranchs exhibit fundamental vertebrate characteristics, including a developmental neural crest, jaws and teeth, and an adaptive immune system. They also are among the oldest existing vertebrates with a closed, pressurized circulatory system and related signaling molecules. The human genome and other vertebrate sequencing projects have underscored the importance of comparative genomics. In many cases, mammalian sequences have limited comparative value for differentiating between conserved regions that are functionally significant and those that simply lack divergence time. For regions of genomes that evolve more slowly, the need for evolutionarily distant organisms may be particularly important. Because of their evolutionary distance from humans, elasmobranch sequences may play an important role in refining predictions of conserved coding and non-coding regions in the human genome.
Among elasmobranches the little skate (Raja or Leukoraja erinacea) and spiny dogfish shark (Squalus acanthias) are commonly used biomedical models. Elasmobranchs exhibit primitive but powerful processes for dealing with salt and water homeostasis, cell volume regulation, and environmental and internal osmotic sensing. The mechanisms by which humans regulate intra- and extracellular small molecule concentrations are fundamentally the same as those utilized by elasmobranchs. However, the major transporting tissues of sharks and skates are sometimes easier to study than mammalian tissues because some elasmobranch tissues are simpler in organization, more compartmentalized and dedicated to fewer functions. Genomic information applied to existing data in these physiological systems will allow the testing of molecular hypotheses and understanding of regulatory mechanisms that are directly applicable to human biology.
Genomic sequence will also provide critical information for other research areas in which elasmobranch models are used, such as the elucidation of the organization and functional significance of gene families critical in immunology. Considerable effort has been devoted to examination of genes related to adaptive immunity in several species of sharks and the clearnose skate (Raja eglanteria), a close relative of the little skate. The Squaliformes, which contain Squalus acanthias, and Rajiformes, which contain Raja erinacea and eglanteria, shared a common ancestor with the other orders of elasmobranchs 200 million years ago. Squaliformes and Rajiformes shared a common ancestor 150 million years ago, making the little skate and dogfish shark relatively close evolutionarily. Other sharks used experimentally in biomedical research include the spotted dogfish shark (order Carcharhiniformes:Scyliorhinus canicula), sandbar shark (Carcharhinus plumbeus), nurse shark (Order Orectolobiformes: Ginglymostoma cirratums) and horn shark (Order Heterodontiformes: Heterodontus francisci). Bacterial artificial chromosome (BAC) genomic libraries previously have been constructed for horn shark, nurse shark and clearnose skate.
The role of the Mount Desert Island Biological Laboratory
Mount Desert Island Biological Laboratory provides experience and support for studies using little skate and spiny dogfish shark through its Centers for Marine Functional Genomic and Membrane Toxicity Studies. Biomedical research at the Laboratory on the little skate and dogfish shark dates from the 1920’s work of Homer Smith. The Center for Marine Functional Genomic Studies, established in 2001, is unique in focusing on biomedical applications of marine genomics. The Center supports programs in Nucleic Acid Sequencing and Analysis, Comparative Genomics and Bioinformatics, and Marine Organism Cell Lines and Stem Cells. Center Director is John Forrest, M.D., Director of Mount Desert Island Biological Laboratory and Yale University Professor of Medicine. Associate Director is David Barnes, Ph.D., Senior Scientist (dbarnes@mdibl.org). The Center for Membrane Toxicity Studies is supported by the National Institute of Environmental Health Sciences (NIEHS). CMTS was established in 1985 as one of four national Marine and Freshwater Biomedical Sciences Centers, and is under the direction of James Boyer, M.D., Ensign Professor of Medicine and Director of the Yale University Liver Center. The Center for Membrane Toxicity Studies convenes internationally recognized investigators to study cellular and molecular mechanisms of toxicity of environmental pollutants using elasmobranch and other aquatic models. The Laboratory also is the home of the NIEHS-supported Comparative Toxicogenomics Database. Major goals of this program include annotating genes and proteins of biomedical significance and promoting comparative studies of these genes and proteins across evolutionarily diverse organisms with a particular emphasis on aquatic species. It is the intent that these comparisons will lead to better understanding of molecular evolution, the significance of conserved sequences and the genetic basis of variable susceptibility to disease and toxicity.
BAC genomic libraries (4X coverage) for little skate and spiny dogfish shark were completed in January, 2005, in collaboration with the Clemson University Genomics Institute. Greater BAC coverage of the little skate genome will be forthcoming. The Center for Marine Functional Genomic Studies also is engaged in a continuing program of little skate and spiny dogfish shark EST generation and sequence analysis using normalized libraries from a variety of tissues. Thus far, approximately 5,700 little skate and 4,500 spiny dogfish shark sequences have been provided to Genbank/NCBI. Ongoing research at Mount Desert Island Biological Laboratory using the spiny dogfish shark and little skate to understand disease states includes studies of the cystic fibrosis transmembrane regulator, other ion transport proteins, and control of osmolyte regulation. One of the major areas in which the little skate is used at the Laboratory is in the study of hepatic xenobiotic transport and excretion in liver disease and toxicity. This research is supported largely by NIEHS. A program also is devoted to establishing transfection and cell culture technology for little skate and spiny dogfish shark. Once sufficient genomic information is available, in vitro approaches can be used to conduct targeted studies to evaluate functionality of proteins and regulatory regions in the DNA of these organisms. Cell cultures in combination with genomic information will allow physical mapping and construction of radiation hybrid panels. The haploid genome sizes of the little skate and spiny dogfish shark are 3.5 pg/nucleus and 6.5 pg/nucleus. The haploid chromosome number is 49 for little skate and 30 for spiny dogfish shark. The heterozygosity of the little skate is unknown, and preliminary analyses of several animals will be necessary prior to large-scale sequencing.
The Contribution of Skates and Sharks to Improving Human Health and Understanding Disease
The little skate and spiny dogfish shark live in ocean temperatures of 5-10oC, and can be maintained year-round under appropriate laboratory conditions. These cold-water organisms exhibit much reduced rates of metabolism, ion transport and oxygen consumption compared with many animal models. This results in increased stability of cells, tissues and cellular macromolecules, including nucleic acids, and more accessible quantitative physiological measurements, such as membrane transport rates. These animals are sufficiently large to provide plentiful amounts of material for cell culture or biochemistry, and use of the spiny dogfish shark rectal gland as a source of Na/K-ATPase was a critical part of the Nobel Prize-winning work of Dr. J.C. Skou on this enzyme. The experimental accessibility of the little skate and the evolutionary distance from humans has promoted unique insights into conserved functional domains of genes associated with human liver biology, multidrug resistance and xenobiotic transport. Multidrug resistance-associated protein-2 in skate shows high identity to the orthologous human protein in several transmembrane domains known to function in substrate recognition and transport. The bile salt export pump, (BSEP) also has been studied extensively using the cloned skate transporter gene. Mutations in BSEP are involved in a type of liver disease, progressive familial intrahepatic cholestasis. Compared to the human ortholog, skate BSEP exhibited similar substrate specificity, transport function and expression. Sequence comparisons with the skate gene show that all of the disease-related human mutations are in regions conserved between human and skate. Hepatocytes from the little skate may retain hepatobiliary polarity up to several days in culture, offering significant advantages over mammalian hepatocyte culture.
Skates and sharks have interesting properties regarding retinal neurobiology, aging, telomerase expression, stem cells and immortalization. For instance, a region of continual renal regeneration has been identified in the little skate, with new tubules being formed continually through adulthood. Elasmobranchs also have received publicity for providing insights into cancer biology. Although media reports claiming that these animals do not get cancer often are scientifically unsubstantiated, elasmobranchs are sources of antibiotics and angiogenesis inhibitors that may be promising cancer treatments. Genomic analysis will lead to a better understanding of how these compounds are synthesized and interact within the organism and may have important implications on the development of therapeutic treatments, including approaches to treatment of drug-resistant cancers.
Another research area that may expand rapidly with genomic data is the use of the skate embryo in developmental biology. Egg-laying females with stored sperm can be identified by palpation, and can be maintained in tanks for many months while predictably producing eggs in pairs at intervals of about seven days. The rate of embryonic development can be modulated by temperature, and development is slow enough for removal and in vitro culture of embryonic cells and transplantation of altered cells back into an embryo to be feasible. With genomic information, skate embryos would be amenable to transgenics, morpholino or other knock-down approaches and in situ hybridization. Parthenogenesis in elasmobranchs has also been reported and provides even larger potential for genomics-based studies. The little skate could be exploited as a model of vertebrate development offering new perspectives in comparative studies with the more traditional developmental models.
Selected Recent Citations: Biomedical Research with Skates.
Anderson MK, Pant R, Miracle AL, Sun X, Luer CA, Walsh CJ, Telfer JC, Litman GW, Rothenberg EV. Evolutionary origins of lymphocytes: ensembles of T cell and B cell transcriptional regulators in a cartilaginous fish. J Immunol. 2004 May 15;172(10):5851-60
Anderson MK, Shamblott MJ, Litman RT, Litman, GW. Generation of immunoglobulin light chain gene diversity in Raja erinacea is not associated with somatic rearrangement, an exception to a central paradigm of B cell immunity. J Exp Med, 1995;182: 109-119.
Ballatori N, Hager DN, Nundy S, Miller DS, Boyer JL.Carrier-mediated uptake of lucifer yellow in skate and rat hepatocytes: a fluid-phase marker revisited. Am J Physiol. 1999 Oct;277(4 Pt 1):G896-904
Ballatori N, Rebbeor JF, Connolly GC, Seward DJ, Lenth BE, Henson JH, Sundaram P, Boyer JL. Bile salt excretion in skate liver is mediated by a functional analog of Bsep/Spgp, the bile salt export pump. Am J Physiol Gastrointest Liver Physiol, 2000;278:G57-63.
Cai SY, Soroka CJ, Ballatori N, Boyer JL.Molecular characterization of a multidrug resistance-associated protein, Mrp2, from the little skate. Am J Physiol Regul Integr Comp Physiol, 2003;284:R125-130.
Cai SY, Wang L, Ballatori N, Boyer JL. Bile salt export pump is highly conserved during vertebrate evolution and its expression is inhibited by PFIC type II mutations. Am J Physiol Gastrointest Liver Physiol, 2001;281:G316-322.
Cai SY, Wang W, Soroka CJ, Ballatori N, Boyer, JL. An evolutionarily ancient Oatp: insights into conserved functional domains of these proteins. Am J Physiol Gastrointest Liver Physiol, 2002;282:G702-710.
Dranoff JA, O'Neill AF, Franco AM, Cai SY, Connolly GC, Ballatori N, Boyer JL. Nathanson MH. A primitive ATP receptor from the little skate Raja erinacea. J Biol Chem, 2000;275:30701-30706.
Eason DD, Litman RT, Luer CA, Kerr W, Litman GW Expression of individual immunoglobulin genes occurs in an unusual system consisting of multiple independent loci. Eur J Immunol. 2004 Sep;34(9):2551-8.
Elferink RP, Ottenhoff R, Fricker G, Seward DJ, Ballatori N, Boyer J.Lack of biliary lipid excretion in the little skate, Raja erinacea, indicates the absence of functional Mdr2, Abcg5, and Abcg8 transporters. Am J Physiol Gastrointest Liver Physiol. 2004 May;286(5):G762-8. Epub 2003 Dec 30
Elger M, Hentschel H, Litteral J, Wellner M, Kirsch T, Luft FC, Haller H. Nephrogenesis is induced by partial nephrectomy in the elasmobranch Leucoraja erinacea. J Am Soc Nephrol, 2003;14:1506-1518.
Goldstein L, Koomoa DL., Musch, MW. ATP release from hypotonically stressed skate RBC: potential role in osmolyte channel regulation. J Exp Zoolog Part A Comp Exp Biol, 2003;296:160-163.
Grosell M., Wood CM, Walsh PJ. Copper homeostasis and toxicity in the elasmobranch Raja erinacea and the teleost Myoxocephalus octodecemspinosus during exposure to elevated water-borne copper. Comp Biochem Physiol C Toxicol Pharmacol, 2003;135:179-190.
Kipp H, Kinne-Saffran E, Bevan C, Kinne, RK.Characteristics of renal Na(+)-D-glucose cotransport in the skate (Raja erinacea) and shark (Squalus acanthias). Am J Physiol, 1997;273:R134-142.
Koob TJ, Callard IP. Reproductive endocrinology of female elasmobranchs: lessons from the little skate (Raja erinacea) and spiny dogfish (Squalus acanthias). J Exp Zool, 1999;284:557-574.
Koomoa DL, Musch MW, Myers DE, Goldstein L.Expression of the skate (Raja erinacea) AE1 osmolyte channel in Xenopus laevis oocytes: monovalent cation permeability. J Membr Biol. 2004 Mar 1;198(1):23-9.
Molina AJ, Verzi MP, Birnbaum AD, Yamoah EN, Hammar K, Smith PJ, Malchow RP.Neurotransmitter modulation of extracellular H+ fluxes from isolated retinal horizontal cells of the skate. J Physiol. 2004 Nov 1;560(Pt 3):639-57. Epub 2004 Jul 22.
Morgan RL, Ballantyne JS, Wright PA. Regulation of a renal urea transporter with reduced salinity in a marine elasmobranch, Raja erinacea. J Exp Biol, 2003;206:3285-3292.
Morgan RL, Wright PA, Ballantyne, JS. Urea transport in kidney brush-border membrane vesicles from an elasmobranch, Raja erinacea. J Exp Biol, 2003;206:3293-3302.
Musch MW, Koomoa DL, Goldstein L.Hypotonicity-induced exocytosis of the skate anion exchanger skAE1: role of lipid raft regions. J Biol Chem. 2004 Sep 17;279(38):39447-53. Epub 2004 Jul 16.
Perlman DF, Goldstein L.The anion exchanger as an osmolyte channel in the skate erythrocyte. Neurochem Res. 2004 Jan;29(1):9-15.
Rebbeor JF, Connolly GC, Henson JH, Ballatori N. ATP-dependent GSH and glutathione S-conjugate transport in skate liver: role of an Mrp functional homologue. Am J Physiol Gastrointest Liver, 2000;279:G417-425.
Runnegar M, Seward DJ, Ballatori N, Crawford JM, Boyer JL. Hepatic toxicity and persistence of ser/thr protein phosphatase inhibition by microcystin in the little skate Raja erinacea. Toxicol Appl Pharmacol, 1999;161:40-49.
Seward DJ, Koh AS, Ballatori, N. Functional complementation between a novel mammalian polygenic transport complex and an evolutionarily ancient organic solute transporter, OSTalpha-OSTbeta. J Biol Chem, 2003;278:27473-27482.
Steele SL, P. H. Yancey and P. A. Wright, Dogmas and controversies in the handling of nitrogenous wastes: Osmoregulation during early embryonic development in the marine little skate Raja erinacea; response to changes in external salinity The Journal of Experimental Biology 207, 2021-2031,s 2004.
Selected Recent Citations: Comparative Genomics and Elasmobranch Research in Toxicology and Medicine.
Aller SG, Lombardo ID, Bhanot S, Forrest JN Jr. Cloning, characterization, and functional expression of a CNP receptor regulating CFTR in the shark rectal gland. Am J Physiol, 1999;276:C442-449.
Amores A, Tohru Suzuki, Yi-Lin Yan, Jordan Pomeroy, Amy Singer, Chris Amemiya and John H. Postlethwait, Developmental Roles of Pufferfish Hox Clusters and Genome Evolution in Ray-Fin Fish. Genome Research 14:1-10, 2004
Ballatori N, Boyer JL, Rockett, JC. Exploiting genome data to understand the function, regulation, and evolutionary origins of toxicologically relevant genes. EHP Toxicogenomics, 2003;111:61-65.
Ballatori N, Villalobos AR. Defining the molecular and cellular basis of toxicity using comparative models. Toxicol Appl Pharmacol, 2002;183:207-220.
Barnes D. and Collodi P. 2005. Fish Cell Lines and Stem Cells in The Physiology of Fishes, 3rd edition, Evans, D., Claiborne, J.B. eds, CRC Press, in press
Berstein RM, Schluter SF, Shen S, Marchalonis JJ A new high molecular weight immunoglobulin class from the carcharhine shark Carcharhinus plumbeus: implications for the properties of the primordial immunoglobulin. Proc Natl Acad Sci U S A. 1996 Apr 16;93(8):3289-93.
Bhargava P, Marshall JL, Dahut W, Rizvi N, Trocky N, Williams JI, Hait H, Song S, Holroyd KJ, Hawkins MJ. A phase I and pharmacokinetic study of squalamine, a novel antiangiogenic agent, in patients with advanced cancers. Clin Cancer Res, 2001;7:3912-3919.
Chiu CH, Amemiya C, Dewar K, Kim CB, Ruddle FH, Wagner GP. Molecular evolution of the HoxA cluster in the three major gnathostome lineages. Proc Natl Acad Sci U S A, 2002;99:5492-5497.
Choe KP, Evans DH. Compensation for hypercapnia by a euryhaline elasmobranch: effect of salinity and roles of gills and kidneys in fresh water. J Exp Zoolog Part A Comp Exp Biol, 2003;297:52-63.
Cornelius F, Mahmmoud YA. Themes in ion pump regulation. Ann N Y Acad Sci, 2003;986:579-586.
Dooley K. and Zon L.I. 2000. Zebrafish: a model system for the study of human disease. Curr Opin Genet Dev 10:252-256.
Duggan AE, Marie RS, Jr, Callard IP.Expression of SR-BI (Scavenger Receptor Class B Type I) in turtle (Chrysemys picta) tissues and other nonmammalian vertebrates. J Exp Zool, 2002;292:430-434.
Hanrahan, JW., Mathews, CJ., Grygorczyk, R., Tabcharani, JA., Grzelczak, Z, Chang, XB, Riordan, JR. Regulation of the CFTR chloride channel from humans and sharks. J Exp Zool, 1996;275:283-291.
Hazon N, Wells A, Pillans RD, Good JP, Gary Anderson W, Franklin CE. Urea based osmoregulation and endocrine control in elasmobranch fish with special reference to euryhalinity. Comp Biochem Physiol B Biochem Mol Biol. 2003 Dec;136(4):685-700.
Lehrich RW, Aller SG, Webster P, Marino CR,. Forrest JN Jr. Vasoactive intestinal peptide, forskolin, and genistein increase apical CFTR trafficking in the rectal gland of the spiny dogfish, Squalus acanthias. Acute regulation of CFTR trafficking in an intact epithelium. J Clin Invest, 1998;101:737-745.
Mattingly C., Parton A., Dowell L., Rafferty J., Barnes D., 2004, Cell and Molecular Biology of Marine Elasmobranchs: Squalus acanthias and Raja erinacea. Zebrafish, 1:111-120
Mattingly CJ, Colby GT, Forrest JN, Boyer JL. The Comparative Toxicogenomics Database (CTD). Environ Health Perspect, 2003;111:793-795.
Mattingly CJ, Colby GT, Rosenstein MC, Forrest JN, Jr,. Boyer JL. Promoting comparative molecular studies in environmental health research: an overview of the comparative toxicogenomics database (CTD). Pharmacogenomics J, 2004;4:5-8.
Ovcharenko I., Loots G.G., Giardine B.M., Hou M., Ma J., Hardison R.C., Stubbs L. and Miller W. 2005. Mulan: Multiple-sequence local alignment and visualization for studying function and evolution. Genome Res 15:184-194.
Ringholm A, Klovins J, Fredriksson R, Poliakova N, Larson ET, Kukkonen JP, Larhammar D, Schioth HB. Presence of melanocortin (MC4) receptor in spiny dogfish suggests an ancient vertebrate origin of central melanocortin system. Eur J Biochem,2003; 270:213-221.
Skou, JC. Nobel Lecture. The identification of the sodium pump. Biosci Rep, 1998;18:155-169.
Thomas JW, Touchman JW, Blakesley RW, Bouffard GG, Beckstrom-Sternberg SM, Margulies EH, et al. Comparative analyses of multi-species sequences from targeted genomic regions. Nature, 2003;424:788-793.
Walter R.B., Rains J.D., Russell J.E., Guerra T.M., Daniels C., Johnston D.A., Kumar J., Wheeler A., Kelnar K., Khanolkar V.A., Williams E.L., Hornecker J.L., Hollek L., Mamerow M.M., Pedroza A .and Kazianis S. 2004. A microsatellite genetic linkage map for Xiphophorus. Genetics 168:363-372.
Wilson JM, Randall DJ, Vogl AW, Iwama GK. Immunolocalization of proton-ATPase in the gills of the elasmobranch, Squalus acanthias. J Exp Zool, 1997;278:78-86.
Winchell CJ, Martin AP, Mallatt J.Phylogeny of elasmobranchs based on LSU and SSU ribosomal RNA genes. Mol Phylogenet Evol. 2004 Apr;31(1):214-24
Selected Earlier Citations: Skate and Shark Research at Mount Desert Island Biological Laboratory.
Smith, H. W. (1929). The composition of the body fluids of elasmobranchs. J. Biol. Chem. 81, 407-419.
Clark, R. W. and Smith, H. W. (1932). Absorption and excretion of water and salts by the elasmobranch fishes. J. Cell. Comp. Physiol. 1, 131-143.
Marshall-Jr EK. The comparative physiology of the kidney in relation to theories of renal secretion. Physiol Rev 1935;14:33-159,
Smith, H. W. (1936). The retention and physiological role of urea in the elasmobranchii. Biol. Rev. 11, 49-82.
Smith HW. The Kidney. Structure and Function in Health and Disease. Oxford: Oxford University Press, 1951
Schmidt-Nielsen B. Concentrating mechanism of the kidney from a comparative point of view. Am Heart J 1961;62:579-586, .
Maren, T.H., J.A. Rawls, J.W. Burger and A.C. Myers. The alkaline (Marshall's) gland of the skate. Comp. Biochem. Physiol. 10:1-16, 1963.
Boylan, J. W. (1967). Gill permeability in Squalus acanthias. In Sharks, Skates and Rays (ed. P. W. Gilbert, R. F. Mathewson and D. P. Rall), pp. 197-206. Baltimore: Johns Hopkins Press.
Goldstein, L. and Forster, R. P. (1971). Osmoregulation and urea metabolism in the little skate Raja erinacea. Am. J. Physiol. 220, 742-746.
Boylan, J. W. (1972). A model for passive urea reabsorption in the elasmobranch kidney. Comp. Biochem. Physiol. 42A, 27-30.
Deetjen, P. and T.H. Maren. The dissociation between renal HCO3- reabsorption and H+ secretion in the skate, Raja erinacea. Pflugers Arch. 346:25-30, 1974.
Stoff JS, Silva P, Field M, Forrest J, Stevens A, Epstein FH.Cyclic AMP regulation of active chloride transport in the rectal gland of marine elasmobranchs. J Exp Zool. 1977 Mar;199(3):443-8.
Hentschel, H., Elger, M. and Schmidt-Nielsen, B. (1986). Chemical and morphological differences in the kidney zones of the elasmobranch, Raja erinacea, Mitch. Comp. Biochem. Physiol. 84A, 553-557.
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