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Chromodomain Helicase DNA Binding Protein 1 (Chd1) Is Required for Orofacial Development in Xenopus

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Chromodomain Helicase DNA Binding Protein 1 (Chd1) Is Required for Orofacial Development in Xenopus Virginia Commonwealth University VCU Scholars Compass Theses and Dissertations Graduate School 2018 Chromodomain Helicase DNA Binding Protein 1 (Chd1) is required for orofacial development in Xenopus Brent Wyatt Follow this and additional works at: https://scholarscompass.vcu.edu/etd Part of the Developmental Biology Commons © Brent Wyatt Downloaded from https://scholarscompass.vcu.edu/etd/5365 This Dissertation is brought to you for free and open access by the Graduate School at VCU Scholars Compass. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of VCU Scholars Compass. For more information, please contact [email protected]. ©Brent Hardin Wyatt 2018 All Rights Reserved Chromodomain Helicase DNA Binding Protein 1 (Chd1) is required for orofacial development in Xenopus A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University by BRENT HARDIN WYATT B.S. Biology, East Carolina University 2009 M.S. Molecular Biology and Biotechnology 2013 Director: Amanda Jane Dickinson, Ph.D. ASSOCIATE PROFESSOR DEPARTMENT OF BIOLOGY Virginia Commonwealth University Richmond, Virginia May, 2018 ii ACKNOWLEDGEMENTS There are no words that can truly express my gratitude to the following people for all the help and support they have provided during my time here at VCU. My mentor Dr. Amanda Dickinson. She has been a very caring and supportive mentor and has provided me with invaluable advice on research, writing, and presentations. She was a continual source of encouragement and helped me do things I never thought I would be capable of. My committee members Dr. Greg Walsh, Dr. Jim Lister, Dr. Rita Shiang, Dr. Laura Mathies, and Dr. Shirley Taylor for their advice on research and writing. My lab members were like family. Nathalie Houssin is one of the kindest people I have ever met and she taught me many of the techniques that I learned here. I regularly asked Stacey Wahl and Allyson Kennedy twenty questions a day and they never turned me away. Nav Bharathan was always happy to help me troubleshoot experiments and provided a lot of positive encouragement during the stressful period of writing my dissertation and preparing for my defense. Deborah Howton was my best friend here and her support helped me continue to strive to better myself. My family have always been there to support me and I cannot thank them enough for everything they have done for me. My friend Crystal Beckvold. She reminds me not to give up no matter how hard things seem. iii TABLE OF CONTENTS ACKNOWLEDGEMENTS ................................................................................................ii LIST OF TABLES ........................................................................................................... v LIST OF FIGURES..........................................................................................................vi LIST OF ABBREVIATIONS .......................................................................................... viii ABSTRACT ................................................................................................................... xii CHAPTER 1 THE ROLE OF CHROMODOMAIN HELICASE DNA BINDING PROTEIN 1 (CHD1) IN OROFACIAL DEVELOPMENT .................................................................. 1 INTRODUCTION ......................................................................................................... 2 Midface and palate development .............................................................................. 2 Using Xenopus to investigate orofacial development ............................................... 3 Retinoic acid signaling .............................................................................................. 4 Chromodomain helicase DNA binding protein 1 ....................................................... 7 Epigenetics modifiers are important for craniofacial development ............................ 9 METHODS ................................................................................................................. 12 1. Animals ............................................................................................................ 12 2. Table of Genes ................................................................................................. 12 3. Chemical Treatments ....................................................................................... 13 4. Imaging ............................................................................................................ 14 5. Computational analysis of Chd1 gene and protein sequences ........................ 16 6. RT-PCR to detect chd1 expression levels and splicing defects in morphants .. 17 7. In situ hybridization .......................................................................................... 20 8. Morpholino knockdown .................................................................................... 22 9. Measurements and morphometrics .................................................................. 23 10. Alcian blue staining ....................................................................................... 24 11. Flow Cytometry ............................................................................................. 25 12. Immunohistochemistry .................................................................................. 25 iv 13. RARE analysis .............................................................................................. 26 14. Statistical analysis ............................................................................................ 27 RESULTS .................................................................................................................. 28 1. Transcriptional regulators are required for orofacial development ................... 28 2. Chd1 is important for facial development in Xenopus laevis ............................ 31 3. Decreased Chd1 results in changes in orofacial genes and cartilage development ........................................................................................................... 41 4. Decreased Chd1 results in increased cell death .............................................. 43 5. Chd1 cooperates with retinoic acid during orofacial development.................... 45 DISCUSSION ............................................................................................................. 51 Chd1 is required for craniofacial development in X. laevis ..................................... 51 Chd1 is required for neural crest and cartilage development .................................. 53 Reduced Chd1 expression does not affect cell division .......................................... 56 Chd1 loss increases cell death ............................................................................... 58 Chd1 cooperates with retinoic acid to regulate orofacial development ................... 60 CONCLUSIONS AND FUTURE DIRECTIONS ...................................................... 65 TABLES AND FIGURES ........................................................................................... 69 CHAPTER 2 ANALYZING THE CELL CYCLE PROFILE IN XENOPUS LAEVIS EMBRYOS .................................................................................................................... 94 INTRODUCTION ....................................................................................................... 94 METHODS ................................................................................................................. 98 Reagents ................................................................................................................ 98 Equipment .............................................................................................................. 98 Collecting samples .................................................................................................. 99 Dissociating tissues into individual cells ............................................................... 100 Analyzing the cell samples with the flow cytometer .............................................. 102 Using additional software to analyze the cell cycle profile .................................... 105 DISCUSSION ........................................................................................................... 106 FIGURES ................................................................................................................. 107 REFERENCES ............................................................................................................ 110 APPENDIX: DEVELOPING METHODS TO UNCOVER MECHANISMS REGULATED BY CHD1 .................................................................................................................... 124 TABLES AND FIGURES ......................................................................................... 126 VITA ............................................................................................................................ 128 v LIST OF TABLES Table 1.1: Epigenetic modifiers that are necessary for craniofacial development ........ 69 Table 1.2: Primer sequences for qRT-PCR .................................................................. 71 Table 1.3: Genes depleted in the orofacial tissues of embryos with a RAR antagonist induced median cleft ....................................................................................................
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