5 ACKNOWLEDGEMENTS
a paper requires herculean effort and this award has spurred me to hang on to my ideas and projects with much tenacity.
For the continuous encouragement and the camaraderie that I am blessed with, I want to thank friends from the Institute of Medical Biology (IMB), Institute of Molecular and Cell Biology (IMCB), National University of Singapore (NUS) and KI. Lovely ex-EBL colleagues – Steve Ogg, John Common, Declan Lunny, Ildiko Szeverenyi, Ng Kee Woei and Huijia for the awesome support – I was fortunate to be part of the group.
My NUS seniors, Farhana and Yusuf, for being such fantastic role models to me – Thank you! Huge hugs to Xiaohui – for the terrific friendship and trust - I don’t know what I will do without you! The entire army of friends in KI for making my stay in Sweden such a joy – Jimmie, Jamie, Yinghui, Xue Yuan, Sharon, Jens, Michelle, Vithia, Delina, Kai’er, Zhixiong, Junwei, Kayoko and Huimin.
For the insightful discussions and incredulous support – thank you Xingang. My love to Ben Jin for teaching me about zinc finger nucleases and for continuously reminding me not to drink too much coffee (yes, I promise to sate my caffine carving with honey instead!) The rest of the team in IMCB including Tom Carney, Claudia, Zhonghua, Harriet, Yosuke, Asha, Swee Chuan, Ashley, Asha, Wilson, Anjali, Haihan and Ashish.
The University of Sheffield postdocs – Sarah Baxendale, Rosemary Kim, Caroline Parkin and Stone Elworthy for imparting excellent expertise and guidance on numerous zebrafish techniques – Thank you! To the AGA team, thank you for the prompt administrative assistance thoughout my PhD studies.
All my love to my family - my mum, dad and sister, thanks for such belief and support which I find endlessly encouraging and motivating. Mummy and RJ, thanks for holding my hand along this exciting and intriguing journey. Lastly to RJ, thank you for making me laugh when things get tough. I am eternally grateful to be blessed with such love!
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Institutionen för mikrobiologi, tumör- och cellbiologi
The zebrafish as a model to elucidate human diseases and development
AKADEMISK AVHANDLING
som för avläggande av medicine doktorsexamen vid Karolinska Institutet offentligen försvaras i styrelserummet, 03-18A, IMCB
Fredagen den 7 oktober, 2011, kl 15.00
av
Samantha Lin Chiou LEE
Huvudhandledare:
Professor Birgitte Lane Karolinska Institutet
Institutionen för mikrobiologi, tumör- och cellbiologi
Sweden
Institute of Medical Biology, A*STAR, Singapore
Bihandledare:
Professor Philip Ingham
Institute of Molecular and Cell Biology, A*STAR, Singapore
Fakultetsopponent:
Professor Peter Currie Monash University
Australian Regenerative Medicine Institute Australia
Betygsnämnd:
Professor Lorenz Poellinger Karolinska Institutet
Institutionen för cell- och molekylärbiologi Sweden
Professor Sven Pettersson Karolinska Institutet
Institutionen för mikrobiologi, tumör- och cellbiologi
Sweden
Docent Christoph Winkler National University of Singapore Department of Biological Sciences Singapore
Stockholm 2011
ABSTRACT
This thesis explores the zebrafish as a model organism to illuminate our understanding of processes that orchestrate the progression of cancer metastasis and myogenesis.
First, I describe the successful establishment of a cancer metastasis model in the zebrafish, specifically to study intravasation - one of the earliest steps in the metastatic cascade. Fluorescently labeled tumour cell are transplanted into pervitelline space of 2 days post fertilisation (dpf) zebrafish embryos before they are exposed to normoxic or hypoxic conditions, allowing us to study the effect of hypoxia on tumour-induced angiogenesis and metastasis. Hypoxia elicited an enhanced angiogenic response and neovascularisation to the transplanted tumour and escalated the extent of metastasis in the living zebrafish embryo. Loss of function experiments such as vascular endothelial growth factor (VEGF) blockade using a clinically available drug – Sunitinib or VEGF morpholino knockdown attenuated tumour-induced angiogenesis and metastasis. The in vivo metastasis assay in zebrafish delivers numerous unique advantages over conventional in vitro cell-based or biochemical chemotaxis assays. The transparent embryo facilitates the tracking of the entire intravasation process in real time within a living organism, allowing us to explore the dynamic interplay of tumour cells and the environmental cues that drive metastasis. This model can be further employed to discriminate between tumour cells of different metastatic potential and to identify novel factors that present as impetus for the earliest step of the metastatic cascade.
Next, I demonstrate the analysis of one class of motility mutants originally identified in the 1996 Tübingen genetic screen that potentially serve as models for human myopathies and dystrophies. Two of these mutants have previously been cloned and shown to encode proteins involved in muscle fibre attachment whilst a third has been found to encode the molecular chaperone Heat shock protein (HSP) 90. I describe the phenotypic and molecular characterization of another of these zebrafish motility mutants, frozen (fro). I present evidence that the frot027c mutation disrupts the locus encoding the autophagy pathway component Atg10. This analysis implicates Atg10 in the assembly of both skeletal and cardiac muscle fibers suggesting a previously uncharacterized role for the autophagy pathway in this process.
Together, my findings illustrate the utility of the zebrafish as a model organism that complements established mammalian and invertebrate models. The fecundity and amenability of the zebrafish to genetic manipulation together with the rapid development and translucency of its embryos combine to provide a powerful system with which to unravel and inform the underlying mechanisms that govern fundamental biological processes and shed light on our understanding of human development and diseases.
ISBN 978-91-7457-429-6