An in vivo study of GTF2IRD1 function and its contribution to the physical features of Williams Beuren Syndrome Cesar P. Canales Martinez. A thesis in fulfilment of the requirements for the degree of Doctor of Philosophy School of Medical Sciences, Faculty of Medicine UNSW Australia Sydney Australia August 2015 THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet Surname or Family name: CANALES MARTINEZ First name: CESAR Other name/s: PATRICIO Abbreviation for degree as given in the University calendar: PhD School: SCHOOL OF MEDICAL SCIENCES Faculty: MEDICINE Title: An in vivo study of GTF2IRD1 function and its contribution to the physical features of Williams Beuren Syndrome Abstract 350 words maximum: (PLEASE TYPE) Williams-Beuren Syndrome (WBS) is a complex neurodevelopmental genetic disorder caused by a hemizygous deletion involving up to 28 genes on chromosome 7q11.23. Amongst the spectrum of physical and neurological defects of WBS, it is common to find sensorineural hearing loss (SNHL) and a characteristic set of facial features. The gene GTF2IRD1, first discovered in our laboratory, and the adjacent, homologous gene GTF2I, both fall within the WBS deletion region. Haploinsufficiency of the transcriptional regulators they encode is thought to account for the major neurological and craniofacial aspects of the disease. The aim of the work presented here is to drill deeper into the function of GTF2IRD1 by investigating the impact of a targeted Gtf2ird1 mouse deletion on neurological and craniofacial phenotypes using direct physiological and cellular analysis methods. A detailed analysis of Gtf2ird1 expression in the inner ear and the skin was conducted. A comprehensive analysis of hearing capacity in Gtf2ird1 knockout mice was carried out by examining the auditory brainstem response (ABR) and the distortion product of otoacoustic emissions (DPOAE). The role of GTF2IRD1 in the development of facial skin was examined by a detailed investigation of the control of epidermal proliferation, differentiation and barrier function, followed by an RNA-Seq analysis, to identify the molecular mechanisms that underpin the facial abnormalities observed in these mice. The hearing analyses revealed that Gtf2ird1 knockout mice have hypoacusis (a higher hearing threshold) in both assessments, indicating that the principal hearing deficit in the mice can be traced to impairments in the cochlear amplifier. We suggest that similar mechanisms may underpin the SNHL experienced by WBS patients. Analysis of the facial skin in knockout mice revealed that keratinocyte proliferation was augmented, keratinized epidermal layers were disorganized and the barrier function was compromised in facial regions where Gtf2ird1 is normally expressed during development. The RNA-Seq analysis demonstrated that these abnormalities correlate with dysregulated expression of several key factors, including FGF7. These data suggest that GTF2IRD1 is a crucial component of the transcriptional regulation machinery controlling facial skin patterning and differentiation and supports a role for its involvement in the causation of the facial characteristics of WBS. Declaration relating to disposition of project thesis/dissertation I hereby grant to the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or in part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all property rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstracts International (this is applicable to doctoral theses only). 30-08-16 …………………………………………………………… ……………………………………..……………… ……….……………………...…….… Signature Witness Date The University recognises that there may be exceptional circumstances requiring restrictions on copying or conditions on use. Requests for restriction for a period of up to 2 years must be made in writing. Requests for a longer period of restriction may be considered in exceptional circumstances and require the approval of the Dean of Graduate Research. FOR OFFICE USE ONLY Date of completion of requirements for Award: THIS SHEET IS TO BE GLUED TO THE INSIDE FRONT COVER OF THE THESIS ORIGINALITY STATEMENT ‘I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged.’ SIGNED …………………………………………………… CESAR P. CANALES DATE: 30-08-2016 i ACKNOWLEDGEMENTS I want to express my gratitude to my supervisors Dr Stephen Palmer and Prof Edna Hardeman for allowing me to join to their team and for providing with all the tools and resources to ensure the development of top quality research. Edna, being the head of the Cellular and Genetic Medicine Unit (CGMU) and head of the Anatomy department, makes you a very busy person, yet you were always available when I needed any type of guidance. You have been so supportive throughout my PhD and I cannot find enough words to express the level of gratitude and admiration I have toward you. Steve, I am fully indebted to you. Your advice on both research as well as on my career have been priceless, you have been a tremendous mentor for me and I am also thankful for your invaluable feedback and support during the intense writing process of thesis. I was fortunate to be part of the CGMU. This is a wonderful place for young scientists to develop surrounded by great people. Thanks for the advice that I received from the CGMU supervisors Dr Anthony Kee, Dr Thomas Fath and Prof Peter Gunning, I really appreciate the time you all have taken to discuss and enrich my work at some stage. To my fellow HDR students Bassem, Cecilia, Nadia, Veronica, Melissa, Teresa, Bin and Iman; thanks for the friendship and for making the work so much enjoyable. Special thanks to Florence Tomasetig, I very much appreciated your enthusiasm for this project and your willingness to help in any possible way. I would also like to thank to the Translational Neuroscience Facility (TNF), especially to Professor Gary Housley and Dr Ann Wong, who introduced me to the wonderful world of the hearing physiology. Thank you both for the time devoted to this project, for the long sessions of data analysis and manuscript preparation for publication, and also for helping me out in getting ready for conference presentations. Also thanks to Dr Jennie Cederholm and Dr Kristina Forud, who were always available in the TNF lab for providing assistance when I encountered technical difficulties utilising the equipment. Thanks to our collaborators whose contributions made also possible to develop and interpret part of this research. To Marc Wilkins and Susan Corley from the NSW System Biology Initiative, for their help with the RNA-Seq analysis, to Anthony ii Hannan and Monique Howard from the Florey Institute of Neuroscience, for performing the behavioural studies, to Andy Liang from Neuroscience Research Australia for providing assistance with the CLARITY technique in the skin and to Annemiek Beverdam from the Developmental and Regenerative Dermatology Unit for her assistance interpreting some of the skin findings from this project. I would also like to thank to the physiology teaching department, especially to Dr Nicole Marden and Dr Lesley Ulman. Thanks for the opportunity of being part of the teaching team; I really enjoyed this experience that served as a great distraction from the hard work at the bench. Last but not least, I want to thank and dedicate this thesis to Paulina, my wife and lab mate. Thanks for your understanding, patience, unconditional love and for being always there having the right answer to all my questions in life. To my family in Chile: my parents Carlos and Sonia and my brother Victor, for their endless support and encouragement to go through this journey. To my son Diego, who has been the light of my life for the past 3 years bringing the strength that I need to overcome any obstacle in life. I could not have completed my research without the support of all these wonderful people. iii PUBLICATIONS PUBLISHED Canales CP, Wong AC, Gunning PW, Housley GD, Hardeman EC, Palmer SJ. The role of GTF2IRD1 in the auditory pathology of Williams-Beuren Syndrome. Eur J Hum Genet. 2014 doi: 10.1038/ejhg.2014 IN PREPARATION Canales CP, Corley SM, Kaur P, Smyth I, Carmona-Mora P, Beverdam A, Wilkins MR, Hardeman EC, Palmer SJ. GTF2IRD1 controls epidermal cell proliferation and facial skin patterning in development. Corley SM, Canales CP, Carmona-Mora P, Hardeman EC, Wilkins MR, Palmer SJ. RNA-Seq analysis shows Gtf2ird1 knockout affects genes involved in epidermal and neurological development. iv CONFERENCE ABSTRACTS 2012: Analysis of craniofacial and hearing defects in a mouse model of Williams- Beuren syndrome. (Oral Presentation) Cesar P. Canales, Ann C.Y. Wong, Gary D. Housley, Peter W. Gunning, Edna C. Hardeman and Stephen J. Palmer. Australian Society for Medical Research, Medical Research Week NSW Scientific Meeting, Sydney, NSW, Australia. 2012: The role of GTF2IRD1 in the Williams-Beuren syndrome cognitive profile. Hardeman EC, Howard M, Canales CP, Widagdo J, Taylor KM, Carmona-Mora P, Wong AC, Housley GD, Hannan AJ, Gunning PW and Palmer SJ.