The role of sox4 in regulating choroid fissure closure and retina

University of Kentucky UKnowledge Theses and Dissertations--Biology Biology 2016 The Role of Sox4 in Regulating Choroid Fissure Closure and Retinal Neurogenesis Wen Wen University of Kentucky, wen.edu Digital Object Identifier: http://dx.180 Right click to open a feedback form in a new tab to let us know how this document benefits you. Recommended Citation Wen, Wen, "The Role of Sox4 in Regulating Choroid Fissure Closure and Retinal Neurogenesis" (2016). Theses and Dissertations--Biology.edu/biology_etds/35 This Doctoral Dissertation is brought to you for free and open access by the Biology at UKnowledge. It has been accepted for inclusion in Theses and Dissertations--Biology by an authorized administrator of UKnowledge.

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Wen Wen, Student Dr. Morris, Major Professor Dr. Westneat, Director of Graduate Studies THE ROLE OF SOX4 IN REGULATING CHOROID FISSURE CLOSURE AND RETINAL NEUROGENESIS __________________________ DISSERTATION __________________________ A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Arts and Sciences at the University of Kentucky By Wen Wen Lexington, Kentucky Director: Ann C. Morris, Associate Professor of Biology Lexington, Kentucky 2016 Copyright © Wen Wen 2016 ABSTRACT OF DISSERTATION THE ROLE OF SOX4 IN REGULATING CHOROID FISSURE CLOSURE AND RETINAL NEUROGENESIS The development of the vertebrate eye is tightly controlled by precise genetic regulations.

From a single ocular primordium to bilateral eyes with complex structures and cell types, it requires intensive proliferation and migration for cells in both the ectoderm and mesoderm to accomplish ocular morphogenesis, and during this process cell differentiation and interaction takes place to establish the complex composition of ocular cell types and cellular connections. Genetic defects can lead to severe abnormalities in eye morphogenesis and cell differentiation during ocular development. A tremendous amount of work has been done to identify both intrinsic and extrinsic factors that regulate ocular development. However, much more work is needed to fully understand this complex process.

Sox4 is known as a transcription activator that regulates cell survival and differentiation in multiple embryonic tissues during development. Evidence of its requirement during ocular development has recently emerged, but the mechanism by which Sox4 regulates ocular development is far from elucidated. Chapter 1 of this dissertation provides an overview of different stages in embryonic eye development and known genetic interactions during each stage. It also reviews recent knowledge about SoxC proteins and their roles in ocular development.

Chapter 2 presents data characterizing the expression profile of the zebrafish sox4 co-orthologs, sox4a and sox4b, in the developing eye. Additionally, it presents data from morpholino-mediated sox4 knockdown in zebrafish, which indicate that Sox4 deficiency leads to defects in choroid fissure closure through elevation in the Hedgehog (Hh) signaling pathway. Sox4 knockdown causes upregulation of the Hh ligand indian hedgehog b (ihhb), which alters the proximal-distal boundary of the optic vesicle and inhibits choroid fissure closure. Chapter 3 presents data reporting the generation of sox4 mutant zebrafish lines using the CRISPR/Cas9 genome editing system.

Characterization of one sox4a maternal zygotic (MZ) mutant line confirms Sox4’s role in negative regulation of Hh signaling and reveals new evidence that maternal and zygotic sox4 are both critical for ocular development. Chapter 4 presents data demonstrating that sox4 is required for rod photoreceptor neurogenesis. Rod photoreceptor terminal differentiation is delayed in both sox4 morphants and sox4 CRISPR mutants, while rod progenitor and precursor cells are properly specified. In Chapter 5, the roles of Sox4 in regulating ocular development are summarized based on the results, and implications of the results are discussed to expand our understanding of the genetic regulation of ocular morphogenesis and retinal neurogenesis.

KEYWORDS: Sox4, Eye development, Coloboma, Hedgehog signaling, Rod photoreceptor, CRISPR/Cas9 Wen Wen Student’s Signature April 26, 2016 Date THE ROLE OF SOX4 IN REGULATING CHOROID FISSURE CLOSURE AND RETINAL NEUROGENESIS By Wen Wen Ann C. Morris Director of Dissertation David F. Westneat Director of Graduate Studies April 26, 2016 Date To Mama, Baba, and Qi ACKNOWLEDGEMENTS My six years of graduate study was like an adventure in the science ocean that was full of excitement and challenge. I would like to give my sincere thanks to many who were like the lighthouse that guided my direction and gave me tremendous amount of supports throughout my voyage.

First and foremost I would like to convey my deepest gratitude to my doctoral advisor Dr. She is my role model in any aspects of science and life. She sets an incredible example as an excellent scientist, mentor and friend that influences me every day. Ann, thank you for leading me into the fascinating world of zebrafish eye development, thank you for teaching me how to do science, talk science, write science and enjoy science, thank you for encouraging me to explore science freely with your endless supports and guidance, and thank you for walking me through the ups and downs during both my graduate study and personal life.

Your enthusiastic attitude towards life and science is also deeply rooted in my heart that made me believe that one day I can also become a great scientist like you. No word can express my appreciations to you; all in all I would like to say that you are the one true Queen in the seven kingdoms of my scientific world. I would also like to express my gratitude to my doctoral committee members. Vinnie, thank you for sharing your wisdom and broad knowledge with me and thanks for all the things you have done for us from writing great recommendation letters, raising our stipend to holding annual Christmas parties at your house.

Thanks for holding everybody from Biology together as a big family. Bruce, thank you very much for encouraging me and give me insightful suggestions throughout my graduate training. Pete, thank you very much not only for teaching me how to do critical thinking, but also how to pass my passion to science and research to the younger generation through teaching. I will carry your philosophy of learning and teaching for the rest of my scientific career.

JJ, thank you very much for providing me so much priceless advices toward my graduate study and career development. I would also like to thank all the faculties from Biology. I am so grateful for your patience with my millions of questions and generosity for sharing your lab equipment whenever I need to use. Also, I want to thank Dr.

Downie, my outside defense committee member. Thank you so much for editing my dissertation and giving me valuable suggestions to make it better. I would like to thank all past and present Morris lab students. Lakshmi, Marie, Stephen, Hannah, CC, and Becky, thank you all for your friendship and support.

You made my daily life in iii the lab full of joyful memories! You are like my sisters and brothers in the lab. I cannot forget all the ‘crazy’ things and all the good science we have done together. Thanks very much for sharing your scientific ideas and encouragement with me. Sara, thank you so much for taking care of our fishroom and lab business.

You are the most important core of our daily lab business. None of our research will be done smoothly without your hard work and great support! The same appreciation to Tony, our previous ‘fish man’. Also, thanks to my wonderful undergraduate trainee Joanna and Abi. I am so proud of you two.

Your bright and hardworking minds will bring you success whatever future careers you choose. I also offer my sincere gratitude to the members of the academic staff for their support and help. Jacqueline Burke, Jaclyn Gibson, Jacqueline Lee, Michael Adams, Seth Taylor, George White, Beverly Taulbee, Monica Decker, Cheryl Edwards, thank you so much for keeping our department running smoothly so that we students could only focus on our study and research. You make our department a warm family to everyone.

The degree will benefit me for my career, but the friendship I earn will be with me for my entire life. I would like to thank all my friends who I will always love and remember. Qian, Min, Ye, Lina, Lingfeng, Ting, Wenhui, Houfu, Gang, Zheng, Hui, Yuechen, Qingchao, Yuting, Weikai, Dangdang, Shang, Diao, Yifeng, Bingwei, Chanung, Swagata, Deep, thank you for being there for me whenever I need you. I also want to give special thanks to the Nannan family.

They gave me tremendous amount of love and support. They made it home for me ever since the first night I landed in Lexington. Thank you for all the things you have done for me! Finally, I would like to thank my family: mom, dad and my husband Qi. My dissertation is dedicated for them.

Even the most beautiful word cannot express my love to them. Their love and encouragement always accompany with me and make me who I am today. iv TABLE OF CONTENTS ACKNOWLEDGEMENTS. iii TABLE OF CONTENTS.

v LIST OF TABLES. ix LIST OF FIGURES. x CHAPTER 1: KEEPING AN EYE ON SOXC PROTEINS: VERTEBRATE EYE DEVELOPMENT AND SOXC TRANSCRIPTION FACTORS .3 Origin and morphogenesis of the vertebrate eye.1 Optic primordium specification .2 Eye field segregation.3 Optic cup and optic stalk formation .4 Cellular differentiation of the vertebrate eye .1 The anterior segment.2 The retina and the retinal pigmented epithelium (RPE).6 Hedgehog (Hh) signaling and vertebrate eye development .1 Vertebrate Hh signaling transduction .2 The role of Hh in vertebrate optic vesicle patterning .3 The role of Hh during the cellular differentiation of vertebrate eye .7 Zebrafish as a model for studying eye development.8 SoxC and eye development .1 Sox Subfamilies in vertebrates.2 SoxC proteins and their known target genes .3 SoxC expression in the developing vertebrate eye .4 Functional studies of SoxC proteins in animal models during eye development .9 SoxC and human ocular defects .10 Conclusions and perspective .11 Rationale, Hypothesis and Specific Aims. 37 v CHAPTER 2: SOX4 REGULATES CHOROID FISSURE CLOSURE BY LIMITING HEDGEHOG SIGNALING DURING OCULAR MORPHOGENESIS .1 Sox4a and sox4b are expressed in periocular tissues and the developing retina .2 Knockdown of sox4 causes ocular coloboma .3 Proximo-distal patterning of the optic vesicle is altered in Sox4-deficient zebrafish.4 Sox4-deficient retinas display ectopic cell proliferation.5 Hh signaling is elevated in Sox4-deficient zebrafish .6 Sox4 negatively regulates expression of Indian Hedgehog b (ihhb).7 Bmp7 is a potential intermediate between sox4 and ihhb .8 Sox4 and sox11 have overlapping functions in regulating choroid fissure closure .5 Materials and methods .1 Zebrafish strains and maintenance .