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Development of Novel Transgenic Zebrafish

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Development of Novel Transgenic Zebrafish DEVELOPMENT OF NOVEL TRANSGENIC ZEBRAFISH MODELS AND THEIR APPLICATION TO STUDIES ON ENVIRONMENTAL OESTROGENS Submitted by Jon Marc Green to the University of Exeter as a thesis for the degree of Doctor of Philosophy in Biological Sciences, September 2016 This thesis is available for Library use on the understanding that it is copyright material and that no quotation from the thesis may be published without proper acknowledgement. I certify that all material in this thesis which is not my own work has been identified and that no material has previously been submitted and approved for the award of a degree by this or any other University. (Signature) ……………………………………………(Jon Marc Green) 1 Abstract Oestrogenic chemicals have become increasingly associated with health effects in wildlife populations and humans. Transgenic animal models have been developed to understand the mechanisms by which these oestrogenic chemicals alter hormonal signalling pathways and how these alterations can lead to chronic health effects. The use of highly informative transgenic animal models will also result in better use and potential reduction of intact animals used in animal testing in line with the principles of the 3Rs. In this thesis work, two novel oestrogen responsive transgenic zebrafish models have been generated to investigate the effects of oestrogenic chemicals, identify their tissue targets and better understand the temporal dynamics of these responses. Both models express the pigment-free ‘Casper’ (a mutant line lacking skin pigment) phenotype, which facilitate identification of responding target tissues in the whole fish in all fish life stages (embryos to adults). The oestrogen response element green fluorescent (ERE-GFP)-Casper model was generated by crossing an established ERE-GFP line with the skin pigment free Casper line. The model generated is highly sensitive to oestrogenic chemicals, detecting responses to environmentally relevant concentrations of EE2, bisphenol A (BPA), genistein and nonylphenol. Use of the ERE-GFP- Casper model shows chemical type and concentration dependence for green fluorescent protein (GFP) induction and both spatial and temporal responses for different environmental oestrogens tested. A semi-automated (ArrayScan) imaging and image analysis system was also developed to quantify whole body fluorescence responses for a range of different oestrogenic chemicals in the new transgenic zebrafish model. The zebrafish model developed provides a sensitive and highly integrative system for identifying oestrogenic chemicals, 2 their target tissues and effect concentrations for exposures in real time and across different life stages. It thus has application for chemical screening to better direct health effects analysis of environmental oestrogens and for investigating the functional roles of oestrogens in vertebrates. The second model generated was an ERE-Kaede-Casper line developed via crossing of the ERE-GFP-Casper line and a UAS-Kaede line and screening subsequent generations for a desired genotype and homozygous expression of the transgenes. Kaede is a photoconvertible fluorescent protein that initially fluoresces green in colour and can be permanently converted to red fluorescence upon short exposure to UV light. The model has a silenced skin pigmentation and high sensitivity to oestrogenic chemicals comparable with the previously developed ERE-GFP-Casper model. Use of this model has identified windows of tissue-specific sensitivity to ethinyloestradiol (EE2) for exposure during early-life (0-48hpf) and illustrated that exposure to oestrogen (EE2) during early life (0-48hpf) can enhance responsiveness (sensitivity) to different environmental oestrogens (EE2, genistein and bisphenol A) for subsequent exposures during development. These findings illustrate the importance of oestrogen exposure history in effects assessments and they have wider implications for the possible adverse effects associated with oestrogen exposure. 3 Information This PhD thesis includes information and results that have been synthesised for three research papers, two of these papers published and the third paper is in preparation for submission for publication at the time of submitting this thesis. “Transgenic Fish Systems and their Application in Ecotoxicology”. Lee, O; Green, JM; Tyler, CR, Critical Reviews in Toxicology, 2015 This is a critical review of techniques and methods used to generate transgenic zebrafish and medaka models for ecotoxicology assessments and includes a comparative analysis of the models themselves. This paper is included in the appendix in its original format. “High-Content and Semi-Automated Quantification of Responses to Estrogenic Chemicals Using a Novel Translucent Transgenic Zebrafish” Green, JM; Metz, J; Trznadel, M; Lee, O; Takesono, A; Kudoh, T; Owen, S; Tyler, CR, Environmental Science and Technology, 2016 This article documents the generation of the translucent ERE-GFP-Casper model and its application in a novel in vivo screening system for semi- automated identification of oestrogenic chemicals, their target tissues and relative potencies. This paper is included in its original format in Chapter 4 of the thesis. 4 “Early life exposure to ethinylestradiol enhances subsequent responses to environmental oestrogens measured in a novel transgenic zebrafish” Green, JM; Lange, A; Scott, A; Wai, HA; Takesono, A; Brown, AR; Owen, SF; Kudoh, T; Tyler, CR; in preparation for submission to Environmental Health Perspectives, 2016 This paper describes the generation of the translucent ERE-Kaede-Casper model that expresses a photoconvertible Kaede fluorescent protein. The model has been used to identify the dynamics of fluorescence response to oestrogenic chemical exposure and assess for sensitivity to oestrogens for sequential (repeated) exposures during early life. This paper is included in its original format in Chapter 5 of the thesis. 5 Acknowledgements I would like to thank my main supervisor, prof. Charles Tyler, who has been hugely supportive during my PhD whilst also instilling a large amount of belief in me to work independently. Our brainstorming meetings were genuinely one of the highlights of my PhD. I want to say a big thank you to my secondary supervisors as well: Dr Stewart Owen, Dr Tetsu Kudoh and Dr Ross Brown. They have been a huge resource of support and expertise and were always available to answer any questions I may have had. A special mention to Dr Ross Brown for helping me settle into the AstraZeneca Brixham Environmental Laboratory during my placement there, it was very much appreciated. I want to thank Dr Aya Takesono for not only teaching me a number of key skills necessary for the work, but for always making me laugh in the lab. I have been fortunate enough to have worked with a number of fantastic researchers during the project: Dr Okhyun Lee, John Moreman, Dr Jeremy Metz, Dr Maciej Trznadel, Dr Matt Winter, Dr Anke Lange and Aaron Scott. These people have made vital contributions towards this thesis and for that I am very thankful. Special mention to Dr Greg Paull who has overseen the maintenance of the zebrafish lines, has been a great friend and was always available to be nutmegged at Tuesday football. Finally I would like to thank my friends for all their encouragement. Emma, for being so patient with my weekends of work and helping me see the light at the end of the tunnel. Most of all, I want to thank my parents for always believing in me. I hope I have done them proud. 6 Table of contents Title Page and Declaration……………………………………………………………1 Abstract and Paper Information………………………………………………………2 Acknowledgements………………….…………………...……………………………6 Contents………………………………………………………………………………...7 List of General Abbreviations………...……………………………………………..10 CHAPTER 1: GENERAL INTRODUCTION 1.1 THE ENDOCRINE SYSTEM……….…………………………………………..13 1.2 OESTROGEN SIGNALLING………….…….………………………………….14 1.3 EDCs AND OESTROGENIC CHEMICALS…………..……………………….16 1.3.1 Early Life Exposure……………………………………………………18 1.3.2 EE2……………………………………………………………………...19 1.3.3 BPA……………………………………………………………………..20 1.3.4 Nonylphenol……………………………………………………………21 1.3.5 Genistein………………………………………………………………..22 1.3.6 Testing Chemicals for Oestrogenic Activity…………………………23 1.4 TRANGENIC BIOSENSORS…………………………………………………...25 1.5 TRANSGENIC MODEL GENERATION……………………………………….30 1.6 SEEING THE RESPONSE – REPORTER GENES………………………….35 1.7 TRANSGENIC FISH AND ECOTOXICOLOGY………………………………39 1.8 ERE-GFP MODELS……………………………………………………………..43 1.9 SUMMARY……………………………………………………………………….46 1.10 AIMS OF THE THESIS.……………………………………………………….48 7 CHAPTER 2: GENERAL METHODS 2.1 GENERAL APPROACH………………………………………………………...53 2.2. ANIMAL HANDLING………………………………………………………..…..55 2.3 EMBRYO WATER…………………………………………………………….....55 2.4 CHEMICAL PREPARATION……………………………………………………56 2.5 ANAESTHESIA…………………………………………………………………..56 2.6 IMAGING……………………………………...……………………………...…..57 2.7 SCREENING……………………………………………………………………..57 CHAPTER 3: GENERATION OF TRANSGENIC ZEBRAFISH MODELS 3.1 INTRODUCTION.………….…………………………………………………….60 3.2 METHODS………………………………………………………………………..62 3.2.1 Generation of ERE-GFP-Casper…………………………………….62 3.2.2 Generation of ERE-Kaede-Casper…………………………………..67 3.3 RESULTS…………………………………………………………………………72 3.3.1 ERE-GFP-Casper……………………………………………………..72 3.3.2 ERE-Kaede-Casper…………………………………………………...78 3.4 DISCUSSION…………………………………………………………………….81 CHAPTER 4: PAPER 1 - HIGH-CONTENT AND SEMI-AUTOMATED SCREENING SYSTEM (ERE-GFP-CASPER) 4.1 ABSTRACT……………………………………………………………………….87 4.2 INTRODUCTION………………………………………………………………...88 4.3 METHODS………………………………………………………………………..91
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