ALMA MATER STUDIORUM UNIVERSITÀ DEGLI STUDI DI BOLOGNA Facoltà di Scienze Matematiche Fisiche e Naturali Scuola di Dottorato in Scienze Biologiche, Biomediche e Biotecnologiche Dottorato di Ricerca in Biologia e Fisiologia Cellulare Ciclo XXII SSD: BIO/11 FUNCTIONAL GENOMICS AND CELL BIOLOGY OF THE DOLPHIN (Tursiops truncatus): ESTABLISHMENT OF NOVEL MOLECULAR TOOLS TO STUDY MARINE MAMMALS IN CHANGING ENVIRONMENTS Presentata da: Dott.ssa ANNALAURA MANCIA Coordinatore Dottorato: Relatore: Prof.ssa Michela Rugolo Prof.ssa Marialuisa Melli - Esame finale 2010 - ALMA MATER STUDIORUM UNIVERSITÀ DEGLI STUDI DI BOLOGNA Faculty of Science Physiology and Cellular Biology XXII PhD Program SSD: BIO/11 FUNCTIONAL GENOMICS AND CELL BIOLOGY OF THE DOLPHIN (Tursiops truncatus): ESTABLISHMENT OF NOVEL MOLECULAR TOOLS TO STUDY MARINE MAMMALS IN CHANGING ENVIRONMENTS PhD Student: Dr ANNALAURA MANCIA Program Coordinator: Supervisor: Michela Rugolo, PhD Marialuisa Melli, PhD - Final PhD exam 2010 - To my sister, Roberta, once again “Dolphins are ‘non-human persons’ who qualify for moral standing as individuals” Thomas White “Research is what I'm doing when I don't know what I'm doing” Wernher Von Braun ABSTRACT The dolphin (Tursiops truncatus) is a mammal that is adapted to life in a totally aquatic environment. Despite the popularity and even iconic status of the dolphin, our knowledge of its physiology, its unique adaptations and the effects on it of environmental stressors are limited. One approach to improve this limited understanding is the implementation of established cellular and molecular methods to provide sensitive and insightful information for dolphin biology. We initiated our studies with the analysis of wild dolphin peripheral blood leukocytes, which have the potential to be informative of the animal’s global immune status. Transcriptomic profiles from almost 200 individual samples were analyzed using a newly developed species- specific microarray to assess its value as a prognostic and diagnostic tool. Functional genomics analyses were informative of stress-induced gene expression profiles and also of geographical location specific transcriptomic signatures, determined by the interaction of genetic, disease and environmental factors. We have developed quantitative metrics to unambiguously characterize the phenotypic properties of dolphin cells in culture. These quantitative metrics can provide identifiable characteristics and baseline data which will enable identification of changes in the cells due to time in culture. We have also developed a novel protocol to isolate primary cultures from cryopreserved tissue of stranded marine mammals, establishing a tissue (and cell) biorepository, a new approach that can provide a solution to the limited availability of samples. The work presented represents the development and application of tools for the study of the biology, health and physiology of the dolphin, and establishes their relevance for future studies of the impact on the dolphin of environmental infection and stress. Index INDEX INDEX I SUMMARY VII 1. INTRODUCTION 1 1. 1 THE BOTTLENOSE DOLPHIN 3 1.1.1 DESCRIPTION AND TAXONOMY 3 1.1.2 PHYLOGENY AND EVOLUTION 6 1.1.3 ADAPTATION TO THE AQUATIC ENVIRONMENT 8 1.2 BOTTLENOSE DOLPHINS AND THE COASTAL ENVIRONMENT 13 1.2.1 EFFECTS OF ENVIRONMENTAL CONDITIONS 13 1.2.2 EMERGING DISEASE AND PATHOLOGY 16 1.2.3 THE BOTTLENOSE DOLPHIN, SENTINEL FOR THE COASTAL ECOSYSTEM 18 1.3 METHODS TO STUDY BOTTLENOSE DOLPHIN HEALTH 19 1.3.1 WILD DOLPHIN SAMPLE COLLECTION 19 1.3.2 TRADITIONAL APPROACHES TO STUDY DOLPHIN HEALTH 22 1.4 MOLECULAR BIOLOGY OF THE BOTTLENOSE DOLPHIN 24 1.4.1 DOLPHIN IMMUNITY: GENES AND PROTEINS 24 1.4.2 OVERVIEW OF MOLECULAR APPROACHES TO STUDY 27 BOTTLENOSE DOLPHIN HEALTH - I - Index 2. RESULTS AND DISCUSSION 33 PART I. THE DOLPHIN TRANSCRIPTOME 35 2.1 THE DOLPHIN MICROARRAY 35 2.1.1 WILD DOLPHINS RESPONSE TO STRESS 35 2.1.2 THE DIFFERENTIALLY EXPRESSED GENES 38 2.1.3 INTERLEUKIN-8 REAL TIME PCR (qRT-PCR) 42 2.2 MICROARRAYS AND ARTIFICIAL NEURAL NETWORKS 45 2.2.1 WILD DOLPHINS AND THE ENVIRONMENT 45 2.2.2 ARTIFICIAL NEURAL NETWORKS (ANNs) 46 2.2.3 ANNs DISCRIMINATION OF SEX DIFFERENCES IN WILD DOLPHINS 47 2.2.4 ANNs DISCRIMINATION OF WILD DOLPHIN’S GEOGRAPHICAL LOCATIONS 50 2.2.5 WILD DOLPHIN POPULATIONS TRANSCRIPTOMIC SIGNATURE 51 2.3 THE DOLPHIN GENOME SEQUENCING PROJECT 57 PART II. DOLPHIN CELL BIOLOGY 59 2.4 QUANTITATIVE CHARACTERIZATION OF MAMMALIAN CELL LINES 59 2.4.1 QUANTITATIVE METRICS TO CHARACTERIZE A DOLPHIN CELL LINE 60 2.4.1.1 MORPHOLOGICAL ANALYSIS: CELL AREA, ROUNDNESS AND CLUSTERING 61 2.4.1.2 VOLUME DISTRIBUTION AND GROWTH RATE CALCULATION 67 2.4.2 THE DOLPHIN LUNG CELL LINE 73 2.4.3 ANALYSIS OF LUNG ENDOTHELIAL CELL MARKERS 83 2.4.4 ANALYSIS OF LUNG EPITHELIAL CELL MARKERS: ALVEOLAR CELLS AND SURFACTANT PROTEIN B 87 2.5 DEVELOPMENT OF MARINE MAMMAL PRIMARY CULTURES 93 - II - Index 2.6 DEVELOPMENT OF MARINE MAMMAL INDUCED PLURIPOTENT STEM CELLS 95 3. CONCLUSIONS 101 3.1 FUNCTIONAL GENOMICS OF THE DOLPHIN 105 3.2 CELL BIOLOGY OF THE DOLPHIN 107 3.3 FINAL CONCLUSIONS 110 4. MATERIALS AND METHODS 111 PART I. MICROARRAY ANALYSIS 113 4.1 OVERVIEW OF THE DOLPHIN MICROARRAY 113 4.1.1 GENERATION OF CDNA LIBRARIES AND COLLECTION OF ESTs 113 4.1.2 EST COLLECTION, GRIDDING AND REARRAYING 114 4.1.3 TARGETED GENE CLONING 115 4.1.4 cDNA MICROARRAY PRODUCTION 118 4.2 BLOOD SAMPLE COLLECTIONS 118 4.2.1 BLOOD SAMPLES FOR THE STRESS INDUCED STUDY 118 4.2.2 BLOOD SAMPLES FOR THE ARTIFICIAL NEURAL NETWORK STUDY 119 4.3 PREPARATION OF RNA 120 4.3.1 RNA EXTRACTION 120 4.3.2 SYNTHESIS OF LABELED RNA 120 4.4 MICROARRAY HYBRIDIZATIONS 121 4.5 STATISTICAL ANALYSIS OF MICROARRAY DATA 121 4.5.1 STATISTICAL ANALYSIS OF MICROARRAY DATA OF THE STRESS INDUCED STUDY 121 4.5.2 STATISTICAL ANALYSIS OF MICROARRAY DATA FOR THE ARTIFICIAL NEURAL NETWORK STUDY 124 - III - Index 4.5.2.1 DATA PROCESSING VIA BIOCONDUCTOR 124 4.5.2.2 GENE SELECTION VIA ANNs 125 4.5.2.3 MACHINE LEARNING ANALYSIS 125 4.6 REAL-TIME PCR (qRT-PCR) 127 4.6.1 qRT-PCR PROTOCOL 127 4.6.2 qRT-PCR DATA ANALYSIS 127 4.7 DOLPHIN GENOME SEQUENCING PROJECT 128 4.7.1 GENERATION OF cDNA LIBRARIES 128 PART II. DOLPHIN CELL BIOLOGY 130 4.8 CELL CULTURE, FIXING AND STAINING 130 4.8.1 CELLS AND CULTURE CONDITIONS 130 4.8.2 CELLS FIXING AND STAINING 131 4.9 MORPHOLOGY ANALYSIS 131 4.10 CELL VOLUME DISTRIBUTION MEASUREMENTS 133 4.11 DOLPHIN CELLS VIDEO-IMAGING 134 4.12 ANALYIS OF ENDOTHELIAL ANDF EPITHELIAL CELL MARKERS 135 4.12.1 IMMUNOCYTOCHEMISTRY 135 4.12.2 IMMUNOHISTOCHEMISTRY 137 4.12.3 ACETILATED LOW-DENSITY LIPOPROTEIN ASSAY 138 4.12.4 WESTERN BLOT ANALYSIS 138 4.13 DEVELOPMENT OF PRIMARY CULTURES 140 4.13.1 DEVELOPMENT OF PYGMY SPERM WHALE ALVEOLAR TYPE II 140 4.13.2 DEVELOPMENT OF PYGMY SPERM WHALE FIBROBLASTS 142 4.14 MARINE MAMMAL INDUCED PLURIPOTENT STEM CELLS 142 - IV - Index 5. APPENDIX 145 Table A1 Dolphins sampled in the stress induced study and their correlated microarray records. 147 Table A2 Genes showing significant regulation in pre versus post blood samples. 149 Table A3 Dolphins sampled in the ANN study and their correlated microarray records 151 Table A4 List of most significantly differentially regulated genes selected from ANNs for the determination of sex in dolphins from 4 geographic locations. 154 Table A5 List of genes showing highest sensitivity values selected from ANNs for the determination of the location in male (I) and female (II) dolphins. 161 Table A6 List of most significantly differentially regulated genes selected from linear statistical analysis (Bioconductor) for the determination of the location in male (I) and female (II) dolphins. 165 6. REFERENCES 169 7. ACKNOWLEDGEMENTS 187 - V - Index - VI - Summary SUMMARY The development of genetic information, molecular tools and reagents for biomedical research in the dolphin (and marine mammals in general) has been slow. Progress in the field has been weakened by the protected status of these animals, which makes obtaining samples difficult. Classical measures of environmental quality for the marine ecosystem have included assessment of the relative abundance of species, and the levels of pollutants found in the environment and in indigenous organisms. These measures are valuable but they do not provide the most sensitive nor most rapid information on the effects of multiple environmental stressors. Major advances in the knowledge of dolphin biology and the unique adaptations of these animals in response to the marine environment are being made as a result of 1) the development of cell-lines for use in in vitro experiments 2) the production of monoclonal antibodies to recognize dolphin proteins 3) the development of dolphin DNA microarrays to measure global gene expression and 4) the sequencing of the dolphin genome. These new approaches may permit the discovery of new genes and/or functions of the proteins for which they encode. Hence, they may play a central role in understanding the complex and specialized biology of the dolphin with regards to how this species responds to an array of environmental insults. The work presented here describes the development of tools for the study of the biology, health and physiology of the dolphin, Tursiops truncatus, and its response to environmental stress and infection. Advances in the knowledge of the molecular genetics of the dolphin have the potential not only to supplement and greatly expand upon conventional measures of - VII - Summary dolphin health status, but also to enhance the sensitivity and potential value of the dolphin as a sentinel species for the health of the marine environment. As a mammal that lives its entire life in the sea, it acts as an integrator of the stressors present in the marine environment.