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BNC1 Regulates Human Epicardial Heterogeneity and Function

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BNC1 Regulates Human Epicardial Heterogeneity and Function BNC1 REGULATES HUMAN EPICARDIAL HETEROGENEITY AND FUNCTION Sophie McManus St Catharine’s College Department of Clinical Medicine/ Cambridge Stem Cell Institute, Jeffrey Cheah Biomedical Centre Addenbrooke’s Hospital University of Cambridge This dissertation is submitted for the degree of Doctor of Philosophy October 2019 Dedicated to A. i DECLARATION This dissertation is the result of my own work and includes nothing which is the outcome of work done in collaboration except where specifically indicated in the text. It has not been previously submitted, in part or whole, to the University of Cambridge or any other university or institution for any degree, diploma, or other qualification. In accordance with the Department of Clinical Medicine guidelines, this thesis does not exceed 60,000 words. Sophie McManus (MA, MRes) Publications Part of the work presented in this dissertation has either been submitted or published in the following: Gambardella L., McManus S.A., Moignard V., Sebukhan D., Delaune A., Andrews S., Bernard W.G., Morrison M., Riley P.R., Le Novѐre N., Sinha S. BNC1 is a master regulator of human epicardial cell heterogeneity and function. Development, accepted for publication in August 2019. (Development 2019 146: dev174441 doi: 10.1242/dev.174441 Published 13th December 2019.) ii ABSTRACT Name: Sophie McManus Thesis title: BNC1 regulates human epicardial heterogeneity and function The epicardium is a transcriptionally heterogeneous cell layer covering the heart, crucial to correct cardiovascular development. Following epithelial-to-mesenchymal transition (EMT), epicardial cells migrate into myocardium, form coronary smooth muscle cells and cardiac fibroblasts, and instruct cardiomyocytes to proliferate and mature. Adult mammalian epicardium is quiescent, but reactivates post-injury with limited effect. However, in zebrafish and in neonatal mouse, epicardial signalling enables robust cardiac regeneration after myocardial infarction. We hypothesise that manipulating human epicardial function could facilitate heart regeneration, via reactivation of embryonic processes. However, epicardial regulation remains incompletely understood; although understanding epicardial mechanisms could be key to potentially manipulating epicardium for therapeutic benefit. This PhD investigates a candidate transcription factor, Basonuclin 1 (BNC1), in functional regulation of human epicardial models, and identifies this gene as a potential key human epicardial regulator. Epicardial-like cells derived from human pluripotent stem cells (hPSC-epi) were previously used for single-cell RNA sequencing (scRNA-seq) in order to investigate possible human epicardial heterogeneity. This identified two distinct hPSC-epi subpopulations: one high in WT1 expression, the other high in TCF21. Bioinformatic analyses identified BNC1 as a potential key node in the hPSC-epi signalling network, via network inference modelling. BNC1 is a transcription factor known to regulate migration and proliferation in other epithelia. Given our network inference analyses and the literature evidence, I hypothesised that BNC1 would have functional relevance in human epicardium, so aimed to investigate its function in hPSC-epi differentiation and epicardial cell migration, as well as identify its putative epicardial targets. Firstly, scRNA-seq data describing hPSC-epi heterogeneity were validated in primary human foetal epicardium and BNC1 expression was confirmed in human epicardial models. BNC1 was subsequently investigated, both by siRNA- knockdown in hPSC-epi and foetal epicardial explants and inducible knockdown cell lines (siKD). siKD hPSC-epi had over 90% BNC1 reduction and displayed significantly altered expression of canonical epicardial genes WT1 and TCF21: hPSC-epi heterogeneity was thereby lost. Altered hPSC-epi proliferation and viability were also iii observed. siKD hPSC-epi was subsequently used in a simple epicardial EMT model (epi- EMT). siKD epi-EMT displayed impaired migration and pronounced cortical actin localisation. ChIP sequencing and bulk RNA sequencing identified potentially promising BNC1 targets, such as actin-binding protein supervillin, for future investigation. We conclude that BNC1 is a key functional epicardial regulator in vitro, paving the way for in vivo characterisation. The knowledge that manipulating BNC1 regulates epicardial heterogeneity and function may instruct efforts to harness epicardial potential for future therapeutic benefit. iv ACKNOWLEDGEMENTS Firstly, I am extremely grateful to Dr Sanjay Sinha for providing the opportunity to undertake my PhD within his research group. It has been both a rewarding and challenging experience; I have truly appreciated his continual intellectual input, critical evaluation and overarching support throughout the project, which has passed incredibly quickly. Being a member of the Sinha group for the past few years has been a fantastic experience. I would also like to thank Dr Laure Gambardella, an inspirational day-to-day supervisor. I am especially grateful to Laure for her kindness, advice and encouragement (combined with a great sense of humour); I have very much enjoyed the time spent working with her and have learnt a great deal, particularly with regards to experimental design and data analysis. I also extend my sincere thanks to all the Sinha group members and collaborators past and present, and in particular thank the following people: Dr Will Bernard, for a great deal of advice and support in establishing the BNC1 knockdown cell lines; Dr Vincent Knight-Schrijver, for his invaluable work in analysing ChIP-seq data and several interesting discussions; Maura Morrison, who was a wonderful rotation student; Dr Maria Colzani, Dr Aishwarya Jacobs and Ms Ping Ong, for invaluable experimental insights; Dr Hongorzul Davaapil, both for scientific guidance and the ImageJ cell counter macro; Alex Petchey for the work on the BNC1 mice; and both Deborah Passey and Dr Peter Holt for general logistical life-saving. All members of the group have been happy to discuss my work, frequently offering helpful feedback and advice: overall, the Sinha group has provided a wonderful working environment, both in terms of scientific achievement and social atmosphere. Dr Helena Kim also offered regular food for thought and professional insight at events outside the lab. I have greatly appreciated the intellectual input from Dr Helle Jørgensen during regular lab meeting presentations and discussions. I am also thankful to Helle for the time I spent in her group during my rotation project, and for her support in pursuing an early PhD side-project on coronary artery proliferation during embryonic development in the Confetti mouse model. Dr Jenny Harman offered useful advice regarding ChIP experiments, and Annabel Taylor has been a great friend throughout the length of my project, providing plenty of experimental discussion over coffee. Cambridge v Cardiovascular Division members have also provided helpful feedback and critical evaluation of my data following regular divisional presentations. I am thankful to the Vallier lab for the gift of the inducible knockdown vector and quantities of helpful advice (in particular, Dr Alessandro Bertero, Stephanie Brown and Dr Anna Osnato). While both the Sinha and the Vallier groups have recently relocated, I have many good memories of working in the LRM (notwithstanding the occasional building-related hiccup). Thanks also go to the Phenotyping Hub, which offered support for flow cytometry; Gregory Strachan and Peter Humphreys, for their useful advice regarding imaging; and Xiaoling He, who coordinated the supply of human foetal tissues. I’ve enjoyed much support from the Oxford girls, Sophie, Helen and Natalia, and have appreciated their unwavering kindness, honesty and humorous take on life. Further thanks are owed to the irrepressible Barton Road girls Anni, Emma and Katie, for their wonderfully absurd sense of humour and fun, as well as several former and continuing members of St Catharine’s College, whom I thank for many great times around Cambridge. I am naturally extremely grateful to my family, and in particular my parents and grandparents, for offering their valuable perspective and encouragement, as ever. This PhD project was enabled by generous British Heart Foundation funding under grant code FS/14/59/31282, and I have been both grateful and proud to represent such a great charity over the last few years. Lastly, I would especially like to thank my fiancé Alex, for being a constant source of support, positivity and kindness. vi CONTENTS 1. INTRODUCTION ..................................................................................................... 23 1.1 THE HEART ............................................................................................................. 24 1.1.1 Cardiovascular disease .................................................................................. 24 1.1.2 Cardiovascular regeneration after injury ...................................................... 25 1.1.3 Cellular therapy for cardiac regeneration ..................................................... 27 1.2 CARDIOVASCULAR ORIGINS: MESODERM DEVELOPMENT ........................................ 28 1.2.1 Mesoderm specification: genes and signalling .............................................. 28 1.2.2 Cardiac lineage formation ............................................................................. 30 1.3 PROEPICARDIAL FORMATION, SIGNALLING AND MIGRATION .................................
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