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Investigating a Microrna-499-5P Network During Cardiac Development

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Investigating a Microrna-499-5P Network During Cardiac Development Investigating a microRNA-499-5p network during cardiac development Thesis for a PhD degree Submitted to University of East Anglia by Johannes Gottfried Wittig This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with the author and that use of any information derived therefrom must be in accordance with current UK Copyright Law. In addition, any quotation or extract must include full attribution. Principal Investigator: Prof. Andrea Münsterberg Submission Date: 10.05.2019 Declaration of own work Declaration of own work I, Johannes Wittig, confirm that the work for the report with the title: “Investigating a microRNA-499-5p network during cardiac development” was undertaken by myself and that no help was provided from other sources than those allowed. All sections of the report that use quotes or describe an argument or development investigated by other scientist have been referenced, including all secondary literature used, to show that this material has been adopted to support my report. Place/Date Signature II Acknowledgements Acknowledgements I am very happy that I had the chance to be part of the Münsterberg-lab for my PhD research, therefore I would very much like to thank Andrea Münsterberg for offering me this great position in her lab. I especially want to thank her for her patience with me in all the moments where I was impatient and complained about slow progress. I also would like to say thank you for the incredible freedom I had during my PhD work and the support she gave me in the lab but also the understanding for all my non-science related activities. I also would like to say thank you to Grant Wheeler, Tracey Chapman and Tamas Dalmay who supervised the progression of my PhD and were always open for questions. Further I would like to thank Geoffrey Mok, for getting me started in the lab by teaching me relevant techniques, showing me around but more importantly for introducing me to the English culture and making every day funny and sarcastic in the lab. Furthermore, a big thanks goes to Camille Viaut and Timothy Grocott for discussions and technical advice during my experiments. Thank you for teaching me important techniques in the lab and discussing my troubles when experiments went wrong. Another big shout out goes to Leighton Folkes and Simon Moxon the bioinformatic brains at UEA, who helped me relentlessly to solve RNAseq challenges but also were great chaps for nerdy discussions. I also would like to acknowledge Paul Thomas from the imaging facility who always helped when there was trouble with imaging tasks. A special thanks goes to the various members of the Münsterberg, Wheeler and Grocott lab for discussion support and making my time in the UK joyful. Very important and not to forget: I would like to thank a few friends of mine: Gerry Giese, Isabell Weber and Marcus Blohs for helping during my PhD research. All the discussions we had were a great input to improve my work and all the advice about science life was invaluable. Thank you guys for the help! Last but not least, I would like to thank my Mum and Dad, as well as my sisters for all the support during my studies. Specifically, I am very thankful for your patience with me when I had spontaneous ideas about moving somewhere else or going abroad and when I needed advice about my future plans. Thank you very much! I am very lucky to have you. III Abstract Abstract Cardiovascular disease is a major cause of death world-wide, which makes it important to study cardiac development. Research into the heart, the first functioning organ in the body, will provide new knowledge to understand potential causes of different heart conditions. Cardiac maturation is a complex process depending on several different signalling cascades, which are fine-tuned by microRNAs. Alterations in NOTCH1 and ETS1 signalling have been linked to congenital heart defects and diseases. NOTCH1 is vital for cardiac septation and trabeculation, whereas ETS1 is necessary for neural crest cell coordination and proper maturation of the endocardial cushions. Further, both of them are important for inducing epithelial to mesenchymal transition in different tissues. Next generation sequencing of small RNAs has identified several microRNAs (miR-126, miR-499 & miR- 451) upregulated during cushion formation (HH17-20 chicken). This was confirmed by RT-qPCR. Interestingly, one of these microRNAs (microRNA-499-5p) targets a site present in the ETS1 and NOTCH1 3’UTR and microRNA-499-5p mimics repressed expression of a luciferase reporter gene. A cardiac injection procedure was developed in the context of this project to asses differential expression and phenotypes after AntagomiR mediated microRNA-499-5p knockdown in vivo. RNAseq revealed strong modulation of extracellular matrix genes, which are connected to ETS1 and upregulation of a NOTCH signalling cascade that affected cardiac Troponin. Along with these gene expression changes we have observed increased ECM deposition at the sites of endocardial cushions by sectioning hearts and subsequent 3D-reconstruction and volume measurements. Moreover, a reduced heart rate in knockdown animals has been observed which was linked to cardiac contraction defects due to reduced TNNT2 expression which may have caused Ca2+ desensitization. TNNT2 reduction came with reduced TBX3 expression which potentially links the observed defects to the conduction system. Future experiments should strengthen the observed links and specify TNNT2s and TBX3s relation to cardiac conduction. IV Table of Contents Table of Contents Declaration of own work . II Acknowledgements . III. Abstract . IV Table of Contents . V. Figure Index . VIII Table Index . IX Abbreviations . .X 1 Aims of Project . 1 2 Introduction . 2 2 .1 MicroRNAs small but potent regulators . 2 2.1.1 Conservation and species differences . .4 2.1.2 Target identification and experimental validation . .5 2.1.3 MicroRNAs in heart development and disease . .7 2 .2 Investigating the signalling network surrounding microRNA-499 . 8 2.2.1 Origin of microRNA-499 and known functions in heart development . .9 2 .3 Formation and Development of the heart . 11 2.3.1 The endocardial cushions and their path to become valves . 12 2.3.1.1 AVC cushions . 12 2.3.1.2 OFT cushions . 13 2.3.1.3 Signalling networks and origin of endocardial cushions . .14 2.3.2 Additional developmental processes initiating around stage HH17 in chick . .16 2.3.2.1 Atrial Septation . 16 2.3.2.2 Ventricular trabeculation and septation . .17 2.3.2.3 Formation of the epicardial cell layer . 18 2 .4 Chicken as model for developmental research . 18 2 .5 Procedures for embryo manipulation to study the heart . 19 3 Material and Methods . .22 3 .1 3D-Reconstruction . 22 3.1.1 Image acquisition . 22 3.1.2 Image alignment . .22 3.1.3 Segmentation . .23 3.1.4 Volume calculation . .23 3.1.5 3D-Volume projection . 23 3 .2 AntagomiR design . 23 3 .3 Bacterial Transformation . 23 3 .4 Cryosectioning and Sample Preparation . 24 3 .5 Embryo Development . 24 3 .6 Embryo Dissections . 25 3.6.1 For whole mount in situ hybridization (WISH) . .25 3.6.2 For RNA extraction . .25 V Table of Contents 3 .7 Experimental evaluation of putative microRNA targets . 25 3.7.1 Generation of luciferase reporter constructs . 25 3.7.2 PCR mediated mutation of microRNA target sites . 26 3.7.3 Luciferase assay of microRNA sensing constructs . 26 3.7.3.1 Cell culture . 26 3.7.3.2 Transfection . .26 3.7.3.3 Luciferase assay . .27 3 .8 Fluorescence microscopy . 27 3 .9 Gel-electrophoresis . 27 3 .10 Heart Rate Assessment . 27 3 .11 Hematoxylin and Eosin Staining . 27 3 .12 In situ hybridisation (ISH) . 28 3.12.1 cDNA synthesis . 28 3.12.2 Generation of gene specific templates for in vitro transcription (IVT) . 28 3.12.3 In vitro transcription for probe generation . .28 3.12.4 Whole mount in situ hybridisation . 29 3.12.4.1 Hybridization . 29 3.12.4.2 Immunodetection . 29 3.12.4.3 Colour development . 30 3.13 In ovo cardiac microinjections . 30 3 .14 Immunohistochemistry . 31 3 .15 microRNA target selection . 31 3 .16 Northern blot . 31 3 .17 pGEM-T-Easy cloning . 31 3 .18 Polymerase Chain reaction . 32 3 .19 Purification and quantification of nucleic acids . 32 3.19.1 Plasmid DNA purification . 32 3.19.2 RNA and microRNA extraction . 32 3.19.2.1 Non-TRIzol® based extraction . 33 3.19.2.2 TRIzol® based extraction . 33 3.19.3 DNA and RNA quantification . .33 3 .20 RT-qPCR . 33 3 .21 RNAseq and Transcriptome Analysis . 35 3 .22 Western Blot . 35 3.22.1 Protein Isolation and SDS-page . .35 3.22.2 Western transfer . .35 3.22.3 Immunolabeling and Imaging . 36 4 Results . 37 4 .1 Validation of microRNAs of interest during development . 37 4.2 In vitro confirmation of target interaction . 37 4 .3 Development of a technique for in vivo knockdown of microRNAs in the chicken heart . 42 4.3.1 Protocol for cardiac injections . 43 4.3.2 Embryo survival and possible timescales of experiments . 44 VI Table of Contents 4.3.3 Validation of AntagomiR administration . 46 4.3.4 Knockdown confirmation is dependent on extraction method . .48 4 .4 Functional analysis of target microRNA knockdownin vivo ..................... 49 4.4.1 Investigating differential expression of cardiac markers prior to RNAseq by ISH ..50 4.4.1.1 Msh homeobox 1 . 50 4.4.1.2 Neuregulin 1 . 50 4.4.1.3 Mothers against decapentaplegic homolog 6 .
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