The Role of Primary Cilia and Sonic Hedgehog Signalling in Adrenal Development Function

The role of primary cilia and sonic hedgehog signalling in adrenal development function. Cogger, Kathryn The copyright of this thesis rests with the author and no quotation from it or information derived from it may be published without the prior written consent of the author For additional information about this publication click this link. http://qmro.qmul.ac.uk/jspui/handle/123456789/3167 Information about this research object was correct at the time of download; we occasionally make corrections to records, please therefore check the published record when citing. For more information contact [email protected] THE ROLE OF PRIMARY CILIA AND SONIC HEDGEHOG SIGNALLING IN ADRENAL DEVELOPMENT AND FUNCTION Kathryn Cogger A thesis submitted to Queen Mary, University of London for the degree of Doctor of Philosophy August 2012 Centre for Endocrinology William Harvey Research Institute Barts and The London School of Medicine and Dentistry Queen Mary University of London ABSTRACT ABSTRACT Primary cilia are sensory organelles found on most vertebrate cells during interphase. They play key roles in development, cell signalling and cancer, and are involved in signal transduction pathways such as Hh and Wnt signalling. The adrenal cortex produces steroid hormones essential for controlling homeostasis and mediating the stress response. Signalling pathways involved in the process of its development and differentiation are still being identified but include Hh and Wnt, and adrenal development is thus likely to require cilia. I have demonstrated that inhibiting cilia formation, using siRNA targeted to different ciliary components, results in reduced differentiation of the human adrenal carcinoma cell line H295R towards a zona glomerulosa (zG)-like phenotype. These data suggest that primary cilia play a key role in adrenal differentiation, but which signalling pathways are involved still remains unclear. I have also discovered that adrenals from Bardet-Biedl syndrome (BBS) mice, the most prominently studied ciliopathy, have thin capsules, the proposed adrenal stem cell niche, and abnormal histology, while zebrafish embryos injected with morpholinos targeting BBS genes show delayed and reduced expression of ff1b, a marker of interrenal tissue. These data further suggest a role for primary cilia in adrenal development and maintenance. These studies are the foundation for elucidating the role of primary cilia in the development and function of the adrenal gland, and furthering our understanding of adrenocortical development. This promises to lead to improved management of adrenal dysfunction, and demonstrating that adrenal defects are a characteristic of ciliopathies will potentially inform new strategies for patient care. 2 ACKNOWLEDGEMENTS ACKNOWLEDGEMENTS First and foremost I would like to thank my supervisor Dr Peter King for his guidance, support and advice during the course of my PhD. I am especially grateful for the benefit of his experience and suggestions in the preparation of this thesis. I would like to thank Dr Paul Chapple for his help and encouragement throughout my PhD, and Dr Leonardo Guasti for his expertise in laboratory techniques. My gratitude also extends to my panel of advisors, Dr Martin Knight and Professor Mike Philpott, for their useful and valuable suggestions about the project. I thank Dr Vincent Marion and Professor Phil Beales for providing the BBS knockout mice, and Dr Rachel Ashworth and Dr Caroline Brennan for their assistance with the collection of zebrafish data. I would also like to acknowledge Dr Wesley Harrison for his help with the FACS analysis, and the Queen Mary Genome Centre for their sequencing and Taqman qPCR services. Thank you to all my colleagues in the department of Endocrinology for making it a fun and enjoyable place to work, and for the unforgettable memories over the last four years. The work of this thesis would not have been possible without the generous financial support of the Medical Research Council, to whom I am most grateful. Finally I would like to thank my friends for their patience and constant encouragement, and my family for their unconditional love and for being so incredibly supportive. I am particularly appreciative for the help and hard work of my parents over the last 26 years, and I hope that I can make them proud. 3 STATEMENT OF ORIGINALITY STATEMENT OF ORIGINALITY I hereby certify that I am the sole author of this thesis and that to the best of my knowledge, my thesis does not infringe upon anyone’s copyright nor violate any proprietary rights and that any ideas, techniques, quotations, or any other material from the work of other people included in my thesis, published or otherwise, are fully acknowledged in accordance with the standard referencing practices. All data was generated by myself except where stated. This thesis has not been submitted for a higher degree to any other University or Institution. 4 TABLE OF CONTENTS TABLE OF CONTENTS CHAPTER 1: INTRODUCTION ..................................................................................... 18 1.1 The Adrenal Gland ............................................................................................ 19 1.1.1 Steroidogenesis ......................................................................................... 20 1.1.2 The stress system, HPA axis, and RAA system .......................................... 21 1.1.3 Cortisol, aldosterone and adrenal androgens .......................................... 26 1.2 Development of the adrenal glands ................................................................. 27 1.2.1 Organogenesis and zonation..................................................................... 27 1.2.2 Signalling pathways ................................................................................... 29 1.2.3 Adrenocortical stem/progenitor cells ....................................................... 34 1.2.4 Adrenal dysregulation ............................................................................... 36 1.3 Adrenal cell lines .............................................................................................. 39 1.4 Shh signalling .................................................................................................... 41 1.4.1 Hh proteins ................................................................................................ 41 1.4.2 Signalling components .............................................................................. 42 1.4.3 Agonists and Antagonists .......................................................................... 45 1.4.4 Hh signalling in the adrenal ....................................................................... 45 1.5 Primary Cilia ...................................................................................................... 47 1.5.1 IFT .............................................................................................................. 49 1.5.2 Signalling pathways ................................................................................... 50 1.6 Nonmotile Ciliopathies ..................................................................................... 51 1.6.1 Bardet-Biedl Syndrome ............................................................................. 52 1.7 Zebrafish ........................................................................................................... 53 1.7.1 Corticosteroids .......................................................................................... 54 5 TABLE OF CONTENTS 1.7.2 Signalling pathways and transcription factors .......................................... 55 1.7.3 Hh signalling and primary cilia .................................................................. 57 1.8 Aims of the project ........................................................................................... 59 CHAPTER 2: MATERIALS AND METHODS .................................................................. 60 2.1 Cell culture ........................................................................................................ 61 2.1.1 Trypsinisation ............................................................................................ 61 2.1.2 Freezing down cells ................................................................................... 61 2.1.3 Counting cells – Haemocytometer ............................................................ 62 2.2 Differentiation .................................................................................................. 63 2.3 RNA Extraction .................................................................................................. 63 2.3.1 DNase treatment ....................................................................................... 63 2.3.2 Phenol extraction ...................................................................................... 64 2.3.3 RNA precipitation ...................................................................................... 64 2.4 First strand cDNA synthesis .............................................................................. 64 2.5 Polymerase chain reaction ............................................................................... 65 2.5.1 Gel electrophoresis ................................................................................... 66 2.5.2 Primers, probes and siRNA sequences ...................................................... 67 2.5.3 Gel purification .........................................................................................

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