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The Role of Small Leucine-Rich Repeat Proteoglycan Asporin in Early Eye Development

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The Role of Small Leucine-Rich Repeat Proteoglycan Asporin in Early Eye Development THE ROLE OF SMALL LEUCINE-RICH REPEAT PROTEOGLYCAN ASPORIN IN EARLY EYE DEVELOPMENT By Kristin Anneliese Luehders Thesis submitted to University College London for the degree of Doctor of Philosophy 2016 UCL Institute of Ophthalmology Faculty of Brain Sciences 1 DECLARATION I, Kristin Anneliese Luehders, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. 2 ABSTRACT There has been a longstanding research interest in understanding the exact mechanisms underlying the correct development of the eye, with the goal of treating eye disease and blindness. While there have been enormous advances in the field of regenerative medicine, there is still some way to go before these advances can be translated into clinical applications. Very little is currently known about the regulatory mechanisms controlling the very early stages of eye development. While the genes, which regulate eye field development have been well characterised, their induction and mechanism of action, including downstream signalling targets and associated downstream signalling systems, still largely remain to be elucidated. The small leucine-rich repeat proteoglycan (SLRP) family of proteins play important roles in a number of biological events, such as proliferation, growth and differentiation. Class I SLRP Asporin (ASPN) has so far been mainly associated with research relating to cartilage homeostasis, osteoarthritis susceptibility and more recently cancer. In this study, I introduce ASPN as a new important factor in Xenopus laevis early eye development. During frog embryogenesis, ASPN is broadly expressed in the neuroectoderm of the embryo. The overexpression of ASPN causes the induction of ectopic eyes. In contrast, blocking ASPN function with morpholino-oligonucleotides inhibits eye formation, indicating that ASPN is an essential factor for eye development. Detailed molecular analyses revealed that ASPN interacts with insulin growth factor receptor (IGF1R) and is essential for activating the IGF-receptor mediated intracellular signalling pathway. Furthermore, ASPN perturbed the Wnt, BMP, and Activin signalling pathways, suggesting that ASPN thereby creates a favourable environment in which the IGF signal can dominate. ASPN is thus a novel secreted molecule critical for eye induction through the coordination of multiple signalling pathways. 3 PUBLICATION LUEHDERS, K.*, SASAI, N.*, DAVAAPIL, H., KUROSAWA-YOSHIDA, M., HIURA, H., BRAH, T. and OHNUMA, S., 2015. The small leucine-rich repeat secreted protein Asporin induces eyes in Xenopus embryos through the IGF signalling pathway. Development (Cambridge), 142(19), pp. 3351-3361. * These authors contributed equally to this paper 4 ACKNOWLEDGEMENTS Firstly, I would like to thank my supervisor Prof. Shin-ichi Ohnuma, for giving me the opportunity to undertake my PhD studies in his lab and also for his support and guidance. I would also like to thank the members (previous and present) of the Ohnuma laboratory. A special big thank you goes to Dr. Noriaki Sasai, who stood both like a mentor and friend to me. His invaluable advice, support and seemingly endless patience when discussing the project and teaching new techniques, helped me to successfully drive this project forward and I will be forever grateful to him. In addition, I would like to thank Dr. Vasiliki Papadaki and Dr. Margaret Dellett for their friendship in and outside the lab and for all the emotional support they provided. My gratitude also goes to my secondary supervisor Prof. Roberto Mayor, who offered encouragement and helpful advice, when I really needed it. To Dr. Barbara Vacca; thanks for sharing the highs and (many) lows of being a Xenopus researcher and for continuously working with me to keep our temperamental frog population happy, so we could carry on with our experiments. Thank you also to my other friends at the Institute: Almu, Rosellina, Lucy, Ewa, Joanna, Viv and Shannon – our lunch dates were always a welcome distraction and made coming to work a lot more enjoyable. Finally, thank you to all my friends outside the lab for being so understanding and for patiently listening to all my “lab chat”. A massive thank you to my partner in life and crime Kieran, for being so patient with me and for always finding a way to motivate me and make me smile. Last but not least, a huge thank you to my loving Mum and Dad, and my brother Daniel, for believing in me and supporting me always. 5 TABLE OF CONTENTS DECLARATION ........................................................................................................ 2 ABSTRACT ................................................................................................................ 3 PUBLICATION .......................................................................................................... 4 ACKNOWLEDGEMENTS ....................................................................................... 5 1 CHAPTER 1: GENERAL INTRODUCTION ................................................ 16 1.1 PREFACE ....................................................................................................... 17 1.2 INTRODUCTION TO THE VISUAL SYSTEM ...................................................... 18 1.2.1 Structure and evolution of eyes ............................................................... 18 1.2.2 Xenopus laevis as a model organism – a historical perspective ............ 23 1.2.3 Xenopus laevis is a great model organism to study eye development .... 24 1.3 EARLY EMBRYONIC DEVELOPMENT ............................................................. 26 1.3.1 Primary embryonic induction and neural induction ............................... 27 1.3.2 Induction of the organizer ....................................................................... 27 1.3.3 Neural induction and the role of BMP inhibitors ................................... 29 1.3.4 Regional specificity of neural induction and axes formation ................. 31 1.4 DEVELOPMENT OF THE EYE AND VISUAL SYSTEM ....................................... 33 1.4.1 Vertebrate eye development .................................................................... 33 1.4.1.1 Eye field specification ..................................................................... 33 1.4.1.2 Neural retina, lens and optic cup development ............................... 34 1.4.1.3 Vertebrate lens, cornea and retina differentiation ........................... 36 1.4.2 Xenopus eye development ....................................................................... 37 1.4.3 Eye field transcription factors in Xenopus .............................................. 39 1.4.3.1 Otx2 (orthodenticle homeobox 2) ................................................... 41 1.4.3.2 Six3 (six homeobox 3) .................................................................... 41 1.4.3.3 Rx1 (retinal homeobox 1) ............................................................... 41 1.4.3.4 Pax6 (paired box 6) ......................................................................... 42 1.4.3.5 ET (eye T-box) ................................................................................ 43 1.4.3.6 Six6 (six homeobox 6) .................................................................... 43 1.4.4 Mechanisms underlying the EFTFs regulation of eye development ....... 44 1.5 EXTRACELLULAR MATRIX AND ROLE OF SLRPS ........................................ 47 1.5.1 Proteoglycans ......................................................................................... 49 1.5.1.1 Intracellular proteoglycans .............................................................. 50 1.5.1.2 Cell surface proteoglycans .............................................................. 50 6 1.5.1.3 Pericellular and basement membrane zone proteoglycans .............. 51 1.5.1.4 Extracellular proteoglycans ............................................................. 51 1.5.2 Small leucine-rich proteoglycans (SLRPs) ............................................. 51 1.5.2.1 Structure and Function .................................................................... 51 1.5.2.2 Role of SLRP in matrix assembly ................................................... 54 1.5.2.3 Diseases linked to SLRPs ................................................................ 55 1.5.2.4 Signalling pathways activated by SLRPs ........................................ 56 1.5.2.5 The role of SLRPs during development .......................................... 56 1.5.3 Asporin – a novel class 1 SLRP .............................................................. 58 1.5.3.1 Identification and characterisation of Asporin ................................ 58 1.5.3.2 ASPN – known signalling properties and disease implications ...... 59 1.6 THE ROLE OF INSULIN-LIKE GROWTH FACTOR (IGF) SIGNALLING IN DEVELOPMENT ....................................................................................................... 61 1.6.1 Overview of the IGF signalling system ................................................... 62 1.6.1.1 IGF1 and IGF2 ................................................................................ 62 1.6.1.2 IGF binding proteins ....................................................................... 63 1.6.1.3 IGF1R and IGF2R ........................................................................... 63 1.6.1.4 IGF signalling through the IGF1R .................................................. 65 1.6.1.5 IGF signalling
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