EXAMINING THE POST-TRANSCRIPTIONAL REGULATION OF LUNATIC FRINGE (Lfng) IN THE MOUSE SEGMENTATION CLOCK DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Kanu Wahi Graduate Program in Molecular Genetics The Ohio State University 2016 Dissertation Committee: Dr. Susan Cole, Advisor Dr. Keith Slotkin Dr. Robin Wharton Dr. Dawn Chandler Copyright by Kanu Wahi 2016 Abstract Somitogenesis is a developmental process in vertebrates involving periodic formation of somites that bud from an unsegmented region known as the pre-somitic mesoderm (PSM) and give rise to the axial skeleton and skeletal muscle in the developed organism. The process of somitogenesis is regulated by a "segmentation clock" that times somite formation and is evolutionarily conserved among vertebrates. Genes, such as Lunatic fringe (Lfng), are required for normal clock function in chickens, mice and humans and exhibit rapid cyclic expression in the PSM with a period that matches the rate of somite formation. To maintain rapid oscillations, especially in the posterior PSM, it is hypothesized that the Lfng transcript should be promptly degraded to ensure its clearance of from cells before the next round of oscillation begins. The 3’UTR of Lfng contains number of conserved sequences that could regulate Lfng expression at the post transcriptional level. In this study we explore the post- transcriptional regulation of the Lfng transcript by the Lfng 3’UTR in the context of the segmentation clock. The Lfng 3'UTR has binding sites for microRNAs (miRNAs), such as miR-125a and miR-200b that are known to affect transcript stability via the 3’UTR. We tested the effect of mutating the mir-125a binding sites in the mouse Lfng 3’UTR on mRNA stability ii of Venus reporter constructs in transgenic mice and observed constitutive Venus expression in the posterior PSM, as opposed to the oscillatory Venus expression when the miR-125a binding sites are not mutated. This implies that the miR-125a binding sites in the Lfng 3'UTR influence RNA turnover in the posterior region of the mouse PSM. Interestingly, when we generated mutant mouse lines that do not express miR-125a we found no effect on somitogenesis and skeletal formation. This indicates that either a compensatory mechanism may be in effect or that miR-125a does not play a role in the mouse segmentation clock. We also examined if another highly conserved region of the Lfng 3'UTR, that is 120 bp in length and has a miR-200 binding site within it, can sufficiently destabilize the transcript. We examined this in two contexts. We found that blocking the miR-200b binding site in the Lfng 3’UTR in chicken embryos has no effect on somitogenesis. Similarly, in mouse myoblast cells the 120 bp sequence of the mouse Lfng 3'UTR is not sufficient to destabilize the transcript. Our results indicate that the 120 bp sequence and the miR-200b binding site within it, are not sufficient to destabilize the transcript and it is likely that this regulation operates in a tissue specific manner. Future work in the lab will examine the function of the endogenous miR-125a binding site in the Lfng 3'UTR on mouse segmentation and skeletal formation. Understanding the post transcriptional regulation of clock genes like Lfng would shed light on mechanisms that maintain oscillatory gene expression in the PSM and ensure timely somite formation. iii Dedication This document is dedicated to my husband, Dhrupad Siddhanta, for his endless support and for being my cheerleader for life. iv Acknowledgments I would like to sincerely thank my advisor, Dr. Susan Cole, without whose constant guidance, motivation and support I would not have been able to make it this far. I would like to acknowledge her patience in bearing with my shortcomings and helping me overcome them. Her nurturing mentorship has made my graduate school experience truly enriching and enjoyable. I would like to thank my lab members: Dr. Maurisa Riley, who provided the stepping stone for this project; Sophia Friesen, an extremely dedicated and hardworking undergraduate student, who has contributed immensely to the analysis of the miR-125a mutant mice; Dr. Dustin Williams, Skye Bochter and Kara Braunreiter for always supporting me and for making lab feel like a second home; Undergraduates, Madeline Parker and Ben Schott for their assistance with lab chores. I am grateful to my committee members: Dr. Dawn Chandler, Dr.Keith Slotkin and Dr. Robin Wharton for sharing their expertise and for their valuable advice during committee meetings that has greatly influenced my project. I would like to thank Dr. Jared Talbot and Dr. Sharon Amacher for sharing their expertise and equipment for the High Resolution Melt Analysis (HRMA). Dr. Vincenzo Coppola, Director of the Mouse facility at OSU for helping us generate the Venus v transgenic and miR-125a mice using the CRISPR/Cas9 system. I immensely thank the all members of the Dr. Harold Fisk’s for always being so helpful, especially Dr. Dwitiya Sawant for guiding me with the use of the fluorescent microscope. I would like to thank the Pelotonia Foundation for their financial support from August 2014 to 2016 and the Alumni Grants Graduate Research Scholarships (AGGRS) association at OSU for funding a part of my thesis project. Lastly, I would like to thank my family: my parents for their unconditional love and support that has helped me reach my goals, my brother and sister-in-law for their encouragement throughout graduate school and my husband for always being by my side and motivating me throughout my graduate career. vi Vita 2003 ...............................................................National Public School, INDIA 2006 ...............................................................B.S. Biotechnology, Kasturba Medical College, INDIA 2008 ...............................................................M.S. Medical Biotechnology, Manipal University, INDIA 2010 to present .............................................Graduate Fellow, Department of Molecular Genetics, The Ohio State University Publications 1. Wahi K, Bochter MS, Cole SE. The many roles of Notch signaling during vertebrate somitogenesis. Semin Cell Dev Biol. 2016 Jan; 49:68-75. Epub 2014 Dec 4. 2. Korlimarla A, Prabhu JS, Anupama CE, Remacle J, Wahi K, Sridhar TS. Separate Quality-Control Measures Are Necessary for Estimation of RNA and Methylated DNA from Formalin-Fixed, Paraffin-Embedded Specimens by Quantitative PCR. J Mol Diagn. 2014 Mar; 16(2):253-60. vii 3. Riley MF, Bochter MS, Wahi K, Nuovo GJ, & Cole SE, mir-125a-5p-mediated Regulation of Lfng is Essential for the Avian Segmentation Clock. Dev Cell. 2013 Mar 11; 24(5):554-61. 4. Prabhu JS, Wahi K, Korlimarla A, Correa M, Manjunath S, Raman N, Srinath BS, Sridhar TS. The epigenetic silencing of the estrogen receptor (ER) by hypermethylation of the ESR1 promoter is seen predominantly in triple-negative breast cancers in Indian women. J Tum Bio. Apr 2012; 33(2). Fields of Study Major Field: Molecular Genetics viii Table of Contents Abstract ................................................................................................................................ii Dedication ........................................................................................................................... iv Acknowledgments................................................................................................................ v Vita ..................................................................................................................................... vii List of Tables ..................................................................................................................... xiii List of Figures .................................................................................................................... xiv Chapter 1: Introduction ..................................................................................................... 1 1.1 Segmentation in vertebrates…………………………………………………………………………………. 2 1.2 The clock and wavefront model can explain periodic somite formation …………………6 1.3 The segmentation clock operates in the vertebrate PSM ……………………………………….7 1.4 Notch signaling pathway establishes cell-to-cell communication ………………………….10 1.5 Notch pathway is required for normal segmentation clock function …………………….14 1.5.1 Role of Notch pathway in the zebrafish segmentation clock……………………….. 17 1.5.2 Role of the Notch pathway in the chicken and mouse segmentation clock…..19 1.6 Setting up the wavefront in the vertebrate PSM………………………………………………….. 25 1.7 Somite patterning in the anterior PSM is influenced by the Notch pathway…………..29 1.8 Fine tuning oscillatory Notch signaling in the segmentation clock………………………….33 ix Chapter 2: Regulation of Lunatic fringe by mir-125a in the mouse segmentation clock 2.1 Introduction………………………………………………………………………………………………………….. 37 2.2.1 Segmentation in vertebrates……………………………………………………………………….. 38 2.2.2 Oscillatory Lunatic fringe expression is essential for the mouse segmentation clock………………………………………………………………………………………………………………39 2.2.3 Post-transcriptional regulation of Lfng by the 3’ UTR…………………………………… 40 2.2 Materials and Methods 2.2.1 Construction of Venus reporter plasmids…………………………………………………….. 45 2.2.2 Transient transfections and generation of stable cell lines………………………..…..47 2.2.3 RT-PCR and Quantitative RT-PCR………………………………………………………………….. 49 2.2.4 Venus transgenic mice………………………………………………………………………………….