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Regulation of Notch Activation by Lunatic Fringe During REGULATION OF NOTCH ACTIVATION BY LUNATIC FRINGE DURING SOMITOGENESIS DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Dustin Ray Williams Graduate Program in Molecular Genetics The Ohio State University 2014 Dissertation Committee: Susan Cole, Advisor Heithem El-Hodiri Mark Seeger Amanda Simcox Copyright by Dustin Ray Williams 2014 ABSTRACT During somitogenesis, paired somites periodically bud from the presomitic mesoderm (PSM) located at the caudal end of the embryo. These somites will give rise to the axial skeleton and musculature of the back. The regulation of this process is complex and occurs at multiple levels. In the posterior PSM, Notch activity levels oscillate as part of a clock that controls the timing of somite formation. In the anterior PSM, the Notch pathway is involved in somite patterning. In the clock, cyclic Notch activation is dependent upon periodic repression by the glycosyltransferase Lunatic fringe (LFNG). Lfng mRNA levels cycle over a two-hour period in the clock, facilitating oscillatory Notch activity. Lfng is also expressed in the anterior PSM, where it may regulate Notch activity during somite patterning. We previously found that mice lacking overt oscillatory Lfng expression in the posterior PSM (Lfng∆FCE) exhibit abnormal anterior development but relatively normal posterior development, suggesting distinct requirements for segmentation clock activity during the formation of the anterior skeleton compared to the posterior skeleton and tail. To further test this idea, we created an allelic series that progressively lowers Lfng levels in the PSM. We find that further reduction of Lfng expression levels in the PSM does not increase disruption of anterior development. However tail development is increasingly compromised as Lfng levels are reduced, suggesting that primary body formation is more sensitive to Lfng dosage than is secondary body formation. Further, we find that low levels of oscillatory Lfng are expressed in the posterior PSM of Lfng∆FCE mutants. This ii reduced expression if sufficient to support relatively normal posterior development, however, we find that the period of the segmentation clock is increased when the amplitude of Lfng oscillations are low. These data support the hypothesis that there are differential requirements for oscillatory Lfng during primary and secondary body formation and that posterior development is less sensitive to overall Lfng levels as long as some oscillatory Lfng is present. For oscillatory Lfng transcription to result in the cyclic Notch activation that is required for somitogenesis, there must be mechanisms in place to rapidly eliminate LFNG protein during the short “off” phase of the clock. We hypothesized that secretion of LFNG is a mechanism for terminating its activity in the clock when it is no longer needed. To test the in vivo relevance of LFNG secretion, we generated knock-in mice expressing a Golgi-tethered LFNG variant that cannot be secreted (LfngtLFNG). Mice carrying a single copy of the LfngtLFNG allele exhibit severe skeletal abnormalities, supporting our hypothesis that tethering LFNG in the Golgi generates a dominant, hyperactive fringe protein that perturbs the segmentation clock. Somites in these mutants do not exhibit proper rostro-caudal patterning and form irregular boundaries. Expression of clock genes is perturbed, and Notch activity levels no longer oscillate. These results support our hypothesis that LFNG processing and secretion play important roles in its function in the segmentation clock, and provide further evidence that post-transcriptional regulation of the segmentation clock is critical during somitogenesis. iii DEDICATION This document is dedicated to my grandmother, for all her love and support. iv ACKNOWLEDGEMENTS I would like to thank my advisor, Susan Cole, for her support, enthusiasm, humor, and her patience. I could not have asked for a more positive work environment. I want to acknowledge previous lab members for their contributions to this work. Emily targeted the mouse mutations used in this study and was a wonderful mentor in the early stages of my graduate career. I’d like to acknowledge Jason Lather for generating the Mesp2>Lfng construct. To Maurisa, thank you for all of your help and support during my graduate studies. I am so fortunate to have been able to work with my closest friend. To Kanu and Skye, thank you for making the lab such an exciting and fun place to work. I wish you all the best v VITA May 8, 1985 ...................................................Born – Fairview, Georgia 2007................................................................B.S. Cellular Biology, University of Georgia 2007 to 2008 ..................................................College of Biological Sciences Dean’s F Fellowship, The Ohio State University 2008 to present ..............................................Graduate Research and Teaching Associate, Department of Molecular Genetics, The Ohio State University PUBLICATIONS Williams DR, Shifley ET, Lather JD, Cole SE. 2014. Posterior skeletal development and the segmentation clock period are sensitive to Lfng dosage during somitogenesis. Dev Biol. 388: 159-169 Williams DR, Shifley ET, Cole SE. Secretion of Lunatic fringe is essential for somitogenesis and segmentation clock function. (In preparation) FIELDS OF STUDY Major Field: Molecular Genetics vi TABLE OF CONTENTS Abstract ............................................................................................................................... ii Dedication .......................................................................................................................... iv Acknowledgements ............................................................................................................. v Vita ..................................................................................................................................... vi Table of Contents .............................................................................................................. vii List of Tables .................................................................................................................... xi List of Figures ................................................................................................................... xii List of Abbreviatinos ....................................................................................................... xiv Chapter 1: Introduction ....................................................................................................... 1 1.1 Introduction ............................................................................................................... 1 1.2 Overview of somitogenesis ....................................................................................... 1 1.3 The clock and wavefront model ................................................................................ 4 1.3.1 The wavefront ..................................................................................................... 5 1.3.2 The segmentation clock ...................................................................................... 7 1.4 The Notch signaling pathway .................................................................................. 11 vii 1.4.1 Overview of Notch signaling ............................................................................ 11 1.4.2 Oscillatory Notch activation is essential for normal segmentation .................. 12 1.4.3 Notch is required for proper somite patterning ................................................ 13 1.5 Post-transcriptional regulation of the segmentation clock ...................................... 15 1.6 Lunatic fringe is essential for oscillatory Notch activation..................................... 18 1.7 Overview ................................................................................................................. 20 1.8 Figures ..................................................................................................................... 21 Chapter 2: Posterior development and the segmentation clock period are sensitive to Lfng dosage during somitogenesis ............................................................................................ 28 2.1 Introduction ............................................................................................................. 28 2.2 Methods ................................................................................................................... 31 2.2.1 Mouse Strains ................................................................................................... 31 2.2.2 Transgenic mouse production and analysis ...................................................... 31 2.2.3 Genotyping ....................................................................................................... 32 2.2.4 Whole mount in situ and immunohistochemistry analysis ............................... 32 2.2.5 Western blot analysis ........................................................................................ 33 2.2.6 Skeletal analysis ............................................................................................... 34 2.2.7 RT-PCR ............................................................................................................ 35 2.3 Results ....................................................................................................................
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