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ELUCIDATING the ROLE of the DAAM PROTEINS in ZEBRAFISH EMBRYONIC DEVELOPMENT a Dissertation Submitted to the Temple University G

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ELUCIDATING the ROLE of the DAAM PROTEINS in ZEBRAFISH EMBRYONIC DEVELOPMENT a Dissertation Submitted to the Temple University G ELUCIDATING THE ROLE OF THE DAAM PROTEINS IN ZEBRAFISH EMBRYONIC DEVELOPMENT A Dissertation Submitted to the Temple University Graduate Board In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY by Carlee M. Cunningham May 2021 Examining Committee Members: Dr. Raymond Habas, Advisory Chair, Department of Biology, Temple University Dr. Darius Balciunas, Department of Biology, Temple University Dr. Karen Palter, Department of Biology, Temple University Dr. Rebecca Burdine, Department of Molecular Biology, Princeton University © Copyright 2021 by Carlee M. Cunningham All Rights Reserved ii ABSTRACT Wnt signaling is an evolutionarily conserved pathway that is essential for the development of the metazoan embryo. Wnt signaling controls essential developmental processes including cell fate, cell polarity, dorsal-ventral patterning and tissue movement (1). Misregulated Wnt signaling can have disastrous effects on the developing human embryo, leading to potentially fatal congenital malformations including anencephaly and spina bifida (2). In addition to embryonic development, misregulated Wnt signaling has been implicated in human pathologies including colon and breast cancers and skeletal malformations (3-5). Wnt signaling is divided into two main pathway branches, canonical or 훽-catenin dependent, and non-canonical, sometimes referred to as the planar cell polarity (PCP) pathway (1). The former branch activates the transcription of the downstream target genes leading to the patterning the dorsal-ventral axis of the developing embryo, whilst the latter has no downstream transcriptional targets but rather acts upon the cytoskeleton to control cell and tissue polarity and movement (6-8). Wnt signaling bifurcates into these two branches at the level of the protein Dishevelled (Dvl) (1, 9). The Dishevelled-associated activator of morphogenesis 1 (Daam1) protein was identified via a yeast-two hybrid screen using Dvl as bait. Daam1 interacts directly with Dvl and mediates activation of the small GTPase Rho, a key player in non-canonical Wnt signaling necessary for proper gastrulation in the Xenopus (frog) embryo (10). In addition to Daam1, vertebrates possess a second Daam, Daam2, originally identified via an in silico screen in humans (11). Similar to Daam1, frog Daam2 iii participates in non-canonical Wnt signaling, contributing to proper formation of the embryonic neural tube. However, conflicting opinions on the function of Daam2 have led to discrepancies regarding its position in Wnt signaling and function in development (12). Daam1/2 have not been extensively investigated at the genetic level, therefore, I employed the genetic model zebrafish (Danio rerio) to further clarify their role in Wnt signaling. Using techniques such as the latest gene-editing system CRISPR/Cas9 and other well-established molecular methods including in situ hybridization, RT-PCR and knockdown using morpholino oligonucleotides, I sought to further establish the role of the Daam protein family in vertebrate embryonic development. Together, my results indicate that the zebrafish Daam1a/b and Daam2 behave similarly to Daam1 and Daam2 in frog, respectively, by participating in the non- canonical Wnt signaling pathway and mediating morphology in the developing zebrafish embryo. iv This dissertation is dedicated to all of the women who are labelled as too loud, too bossy and too aggressive. Keep leading. v ACKNOWLEDGEMENTS The last seven years have been some of the most challenging, exhausting and emotionally taxing of my entire life; they have also been some of the most rewarding, inspiring and fulfilling. I have learned that to achieve a PhD, it takes not only a huge amount of grit and determination, but a village of people behind you, pushing you through the hardest times. The first member of that village for me was Dr. Ray Habas. Coming into graduate school I had no research experience, but Dr. Habas offered me a position in his lab anyway. Although the learning curve was steep, Dr. Habas never gave up on me and continuously encouraged me to keep fighting through the failed experiments and the discouraging results. I cannot thank him enough for taking a leap of faith on me and maintaining that faith over the years. I thank the members of my committee, Dr. Karen Palter, Dr. Darius Balciunas and Dr. Rebecca Burdine for all of their input and constructive criticism. Dr. Balciunas especially was a guiding force during my research and mentored me in a way that was both encouraging yet brutally honest. I cannot thank him enough for investing his time and energy into me and my project despite me not being a member of his lab. I would also like to thank him for offering his lab space when I needed it and sharing his endless knowledge of molecular genetics and zebrafish research with me. I would like to thank all of the previous and current members of the Habas Lab, namely Dr. Noopur Mandrekar, Dr. Baihao Su and Dr. Kaushik Nama. The three of them guided me from the very beginning, from teaching me how to set up a PCR to navigating the NCBI website. They helped form the foundation that I built my research on. A special vi thank you to all the undergrads, particularly Christopher Cassella who allowed me to mentor him for years and helped me with genetic screening and gene cloning. Chris also introduced me to bubble tea from Thai’s and makeup tutorials on YouTube, two things that we enjoyed together during our limited down time on campus. I also extend my thanks to all of the Biology Department faculty and staff. Dr. Waring was always available to offer his seemingly unlimited knowledge of degree requirements and departmental inner workings. During my teaching semesters, Evelyn Vleck and Dr. Dan Spaeth served and wonderful educators who made instructing labs a smooth process, even when teaching over Zoom. Thank you to the Biology administration, Toni Matthews, Tania Stephens, Brenda Mims, Sandy Verma, Carla Woodson and Regee Neely, who keep the department running. Also a huge thanks to our housekeeping staff who ensure our building is sparkling. Finally, an enormous thank you to my family and friends who offered continuous support during this journey. My father, sister and aunt have always served as my cheerleaders, and knew all along that I would accomplish this major goal in my life even when I doubted myself. My mom, although no longer here, instilled in me at a very young age that I was going to have to work hard, aim for perfection, and demand what I needed in life in order to get it. She taught me to never take no for an answer, not even from myself. Last, but certainly not least, I want to thank my partner Christopher. He has only known me as a student, and that title has finally expired! Thank you for never doubting our relationship, sticking with me when our time together was limited to weekends, and being supportive in virtually every way possible. I cannot wait to see where our lives take us. vii TABLE OF CONTENTS Page ABSTRACT ....................................................................................................................... iii DEDICATION .....................................................................................................................v ACKNOWLEDGMENTS ................................................................................................. vi LIST OF TABLES ............................................................................................................. xi LIST OF FIGURES .......................................................................................................... xii CHAPTER 1. INTRODUCTION .........................................................................................................1 History of Wnt signaling ..........................................................................................1 History and function of disheveled ..........................................................................2 Canonical Wnt signaling ..........................................................................................5 Non-canonical Wnt signaling ..................................................................................6 Importance of Wnt signaling in human pathology ................................................11 Actin polymerization, cell migration and neural tube closure ...............................14 Introduction to formins and the Daams .................................................................20 Daam1 in development and disease .......................................................................25 Daam2 in development and disease .......................................................................30 Zebrafish embryonic development ........................................................................35 Techniques for studying gene function in zebrafish ..............................................38 2. MATERIALS AND METHODS .................................................................................41 Sequence conservation studies ...............................................................................41 viii RT-PCR..................................................................................................................41 Cloning ...................................................................................................................42 Whole mount in situ hybridization ........................................................................44 CRISPR/Cas9 mutagenesis ....................................................................................44
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