DETERMINING THE IN VIVO ROLE OF NCK IN ENDOTHELIAL CELLS DURING CARDIOVASCULAR DEVELOPMENT A Thesis Presented to The Faculty of Graduate Studies of The University of Guelph by RICHARD ANDREW HARRIS In partial fulfillment of requirements for the degree of Master of Science October, 2009 ©Richard Harris, 2009 Library and Archives Bibliothfeque et 1*1 Canada Archives Canada Published Heritage Direction du Branch Patrimoine de I'gdition 395 Wellington Street 395, rue Wellington Ottawa ON K1A 0N4 Ottawa ON K1A0N4 Canada Canada Your file Votre reference ISBN: 978-0-494-60393-2 Our file Notre reference ISBN: 978-0-494-60393-2 NOTICE: AVIS: The author has granted a non- L'auteur a accorde une licence non exclusive exclusive license allowing Library and permettant a la Bibliotheque et Archives Archives Canada to reproduce, Canada de reproduce, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par Nnternet, preter, telecommunication or on the Internet, distribuer et vendre des theses partout dans le loan, distribute and sell theses monde, a des fins commerciales ou autres, sur worldwide, for commercial or non- support microforme, papier, electronique et/ou commercial purposes, in microform, autres formats. paper, electronic and/or any other formats. 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Canada ABSTRACT DETERMINING THE IN VIVO ROLE OF NCK IN ENDOTHELIAL CELLS DURING CARDIOVASCULAR DEVELOPMENT Richard Andrew Harris Advisor: University of Guelph, 2009 Dr. Nina Jones Cardiovascular development is a complex process regulated by intercellular signaling mechanisms that coordinate cellular patterning during embryogenesis. The family of Nek adaptor proteins link incoming chemical stimuli at receptor tyrosine kinases on the cell surface to downstream effector molecules that regulate organization of the actin cytoskeleton network. Considering that endothelial cell migration is essential for proper vascular development, and Nek has been shown to regulate this process, we hypothesize that Nek is required for cardiovascular development. Utilizing the Cre-LoxP genetic system, we have demonstrated that deleting Nck2 expression in embryonic endothelial cells of AfcAi-null mice results in lethality between embryonic day 10 and 11. These mice are characterized by specific defects to the vitelline vessel structure of the yolk sac, and the endocardial layering of the primitive heart tube. In order to complement the in vivo mutant phenotype, we have optimized a protocol for isolation of primary endothelial cell cultures from the lungs of Cre", Nckl"7", Nck2flx/flx adult mice, which will be used for further characterization. These results demonstrate the crucial role of Nek in endothelial cells during cardiovascular development, and may serve to further elucidate the signaling mechanisms that underlie this complex process. ACKNOWLEDGMENTS When I first decided to pursue a Master of Science degree at the University of Guelph more than two years ago, I chose my advisor, Dr. Nina Jones, because she seemed smart and understanding, but she was also filled with a spirited and youthful enthusiasm. Looking back on it now, it was her passion for science that drove me to pursue my own goals with that same energy. Over the years, she has helped me to develop as a scientist, but also gave me the freedom to grow as an individual. I owe many successes to her leadership and understanding, and know that she will carry the same enthusiastic pursuit of research for many more years to come. I wish you a full career of reaching your goals, as you have helped me reach mine. I also want to thank Drs. Andrew Bendall and Brenda Coomber for their continual advice and direction as members of my advisory committee. To past and present members of the Jones lab: Steve, Laura, Megan, Melanie, Sylvie, Kelly, Stephanie, Adam, and Colin, thank you for making the lab a fun and enjoyable place to work -1 couldn't have done it without you guys. I owe many thanks to Martha Manning, of the Central Animal Facility, for taking good care of the mice. Dr. Michaela Strueder-Kypke, from the Advanced Analysis Center of the University of Guelph, was invaluable in her help with the confocal microscope. I want to thank Drs. Mira Puri and Aya Kitabayashi, from Mount Sinai Hospital, for their help with the heart sections and letting me use their microscope. I also want to thank Dr. Geoffrey Wood for taking the time to help with the histology and a special thanks goes out to Dr. Reggie Lo for inviting me to play basketball as a break from lab work! I wish to dedicate this work to my wife, Kristel, who continues to be an incredible source of inspiration and energy in my life. With her love and support, I feel that there is nothing I cannot achieve. As you are always there to catch me when I fall, I am no longer afraid to jump. i DECLARATION OF WORK PERFORMED I declare that all work described in this document was performed by myself, with the exception of all items indicated below. Martha Manning, of the Central Animal Facility, performed general maintenance of the mouse colony through feeding and bedding changes, as well as occasionally weaning and ear notching. Histological sectioning and staining was graciously performed by the Advanced Bio-imaging Centre at Mount Sinai Hospital, Toronto. The customer support department at Invitrogen aided in developing the modified protocol for improved endothelial cell selection using Dynalbeads. Dr. Brenda Coomber graciously provided the bEND3 cell line. The Tie2-Cre and Nckl"'", Nck2flx/flx founder lines were generously provided by Dr. Rong Wang (University of California at San Francisco) and Dr. Tony Pawson (University of Toronto), respectively. ii TABLE OF CONTENTS ABSTRACT ACKNOWLEDGMENTS.. ± DECLARATION OF WORK PERFORMED ii TABLE OF CONTENTS iii - v LIST OF TABLES vi LIST OF FIGURES vii LIST OF ABBREVIATIONS viii - ix INTRODUCTION 1 Cardiovascular Development 2 Vasculogenesis in the early mammalian embryo 2 Angiogenesis contributes to maturation of the cardiovascular system 3 Heart development and blood circulation aids in vessel patterning 6 Placenta and extraembryonic membranes 9 Endothelial cell signaling 10 Receptor tyrosine kinase signaling regulates endothelial cell behavior 10 Vascular Endothelial Growth Factor 12 Tie/Tek 12 Platelet-Derived Growth Factor 13 Eph/Ephrin 14 Integrins 15 iii The Nek family of adaptor proteins 17 Introduction to Nek 17 Nek SH3 domains interact with actin-organizing effector molecules 20 RATIONALE AND HYPOTHESIS 23 MATERIALS AND METHODS 25 Objective 1: In vivo characterization of Nek in endothelial cells during development. 25 Animal care policy 25 Transgenic mice housing and care 25 Mouse genetics: Nckl"7", Nck2flx/flx, Tie2-Cre 26 Mouse ear-notch tissue sample preparation for PCR-based genotyping 27 DreamTaq PCR mixture and Thermocycler Conditions 27 Genotype primers 28 Pregnant mouse euthanasia and embryo collection 28 Whole-mount immunofluorescence for yolk sac 29 Histological sectioning and Hematoxylin and Eosin staining 30 Objective 2: In vitro characterization of Nek in cultured endothelial cells 30 Preparation of gelatin/fibronectin coated culture dishes 30 Collagenase treatment of dissected lung tissue 31 Positive sort for endothelial cells 32 Cell immunofluorescence staining 33 iv RESULTS 35 Breeding strategy for generation of Nck-null mouse model 35 Loss of endothelial-Nckresults in embryonic lethality betweenE10 and Ell 37 Thinning of the yolk sac vitelline vessel 42 Defects to the endocardial cushion, myocardial trabeculation and cardiac valves of the heart 46 Primary endothelial cell culture 48 Revised protocol for endothelial cell selection 48 DISCUSSION 52 Loss of endothelial-Nck results in embryonic lethality between E10 and El 1 52 Thinning of the yolk sac vitelline vessel 53 Defects to the endocardial cushion, myocardial trabeculation and cardiac valves of the heart 55 Cardiac output vs. vessel structure 56 Further characterization of the mutant embryo phenotype 58 Primary endothelial cell culture 61 SUMMARY AND CONCLUSIONS 63 REFERENCES 64 v LIST OF TABLES Table Page 1. Primer sequences used for PCR-based genotyping of Cre, Nckl and Nck2 genes 28 2. Endothelial-Nck null pups succumb prenatally 37 3. Loss of endothelial-Nck results in embryonic lethality between E10 and Ell 38 4. Insights into endothelial molecular identity by mouse knockout studies 60 vi LIST OF FIGURES Figure Page 1. Mechanisms of Vasculogenesis and Angiogenesis 5 2. Heart Development 7 3. Endothelial cell signaling pathways contributing vascular development 11 4. Binding partners for Nek 1/2 adaptor proteins 19 5. Breeding strategy for production of endothelial-Nek null mice from Tie2-Cre and Nckl"7", Nck2flx/flx founder lines 36 6. Determining the lethal time-point of mutant embryos 40 7. Enlarged view of embryonic day 10.5 embryos 41 8. Loss of endothelial-Nck results in decreased vessel perfusion on yolk sac 43 9. Loss of endothelial-Nck results in loss of structural integrity of major vitelline vessel of El 0.5 yolk sac 44 10. Recruitment of smooth muscle cells to the yolk sac vitelline vessel 45 11.