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The Regulation of Lunatic Fringe During Somitogenesis

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The Regulation of Lunatic Fringe During Somitogenesis THE REGULATION OF 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 Emily T. Shifley ***** The Ohio State University 2009 Dissertation Committee: Approved by Professor Susan Cole, Advisor Professor Christine Beattie _________________________________ Professor Mark Seeger Advisor Graduate Program in Molecular Genetics Professor Michael Weinstein ABSTRACT Somitogenesis is the morphological hallmark of vertebrate segmentation. Somites bud from the presomitic mesoderm (PSM) in a sequential, periodic fashion and give rise to the rib cage, vertebrae, and dermis and muscles of the back. The regulation of somitogenesis is complex. In the posterior region of the PSM, a segmentation clock operates to organize cohorts of cells into presomites, while in the anterior region of the PSM the presomites are patterned into rostral and caudal compartments (R/C patterning). Both of these stages of somitogenesis are controlled, at least in part, by the Notch pathway and Lunatic fringe (Lfng), a glycosyltransferase that modifies the Notch receptor. To dissect the roles played by Lfng during somitogenesis, we created a novel allele that lacks cyclic Lfng expression within the segmentation clock, but that maintains expression during R/C somite patterning (Lfng∆FCE1). Lfng∆FCE1/∆FCE1 mice have severe defects in their anterior vertebrae and rib cages, but relatively normal sacral and tail vertebrae, unlike Lfng knockouts. Segmentation clock function is differentially affected by the ∆FCE1 deletion; during anterior somitogenesis the expression patterns of many clock genes are disrupted, while during posterior somitogenesis, certain clock components have recovered. R/C patterning occurs relatively normally in Lfng∆FCE1/∆FCE1 embryos, likely contributing to the partial phenotype rescue, and confirming that Lfng ii plays separate roles in the two regions of the PSM. These results reveal that the oscillatory regulation of the Notch pathway plays an important role in the segmentation clock during the development of the anterior skeleton. As part of the segmentation clock, Lfng mRNA is periodically transcribed and LFNG protein levels have also been observed to cycle. Lfng mRNA and LFNG protein molecules must therefore have rapid turnover rates in the PSM. We hypothesize that cyclic Lfng transcription is coupled with signals in the Lfng 3’UTR that confer a short half-life on Lfng mRNA. To test this, we examined the expression patterns of transgenes containing conserved sections of the Lfng 3’UTR and determined which sections cooperatively confer a short RNA half-life in the PSM. LFNG protein acts in the Golgi, but is also cleaved and released into the extracellular space. We hypothesized that this cleavage/secretion contributes to short LFNG intracellular half-life, facilitating its rapid oscillations. To test this, we localized N-terminal protein sequences that control the secretory behavior of the fringe proteins and found that LFNG processing is promoted by furin-like protein convertases. Mutations that alter LFNG processing increase its intracellular half-life in vitro without affecting the specificity of its function in the Notch pathway. To determine the importance of LFNG cleavage/secretion in vivo, we created a novel allele that tethers LFNG to the Golgi (LfngRL). LfngRL/+ mice show significant segmentation and patterning defects suggesting that the short intracellular half-life of LFNG is important for somitogenesis. Thus, the cyclic activity of Lfng in the segmentation clock is achieved through multiple mechanisms, including tight regulation of mRNA and protein levels. Overall, we find that Lfng plays an important role in spatially and temporally regulating Notch signaling during vertebrate segmentation. iii DEDICATION This work is dedicated to my parents, to my siblings, and to my husband for all of their love and support. iv ACKNOWLEDGMENTS I would like to acknowledge my advisor, Dr. Susan Cole, for all of her guidance and encouragement during my graduate studies. I am grateful for the opportunity to have been a part of her research and truly appreciate all of the mentoring and training she has given me. I could not have asked for a more supportive advisor. I would also like to thank my committee members, Dr. Beattie, Dr. Chamberlin, Dr. Seeger, and Dr. Weinstein for their time and guidance during my graduate studies. I am especially grateful to Dr. Weinstein for helping with knock-in mice. I would like to acknowledge current and previous members of the lab for their contributions to this work and their support. Kellie contributed some of the data included in this thesis, as indicated, and was a wonderful co-worker and friend. To the other Cole lab graduate students, Ariadna, Maurisa, and Dustin, thank you for making the lab a supportive and fun place to work, I wish you all the best. To Dawn and Jorge, thank you for your contributions to the GFP transgenic mice and all of the work you do in the lab. To all of the undergraduates who have come through the Cole lab, thank you for your enthusiasm. v I would like to thank my classmates for their camaraderie as we have worked through graduate school together. I have truly enjoyed our friendship and time spent together. I also wish to thank the editors of Birth Defects Research (Part C), Development, and Biochimica et Biophysica Acta: Molecular Cell Research for the use of the previously published text and figures in this dissertation. vi VITA September 29, 1981………………..….Born –Cincinnati, OH. 2003……………………………………Bachelor of Science. Ohio University. 2003 – 2004………………………...….University Fellow The Ohio State University. 2004 – 2009…………………………....Research and Teaching Assistant The Ohio State University. PUBLICATIONS 1. E.T. Shifley and S.E. Cole. (2008) Lunatic fringe protein processing by proprotein convertases may contribute to the short protein half-life in the segmentation clock. Biochim Biophys Acta, Mol Cell Res., 1783.2384-2390. 2. E.T. Shifley, K.M. VanHorn, A. Pérez-Balaguer, J.D. Franklin, M. Weinstein, and S.E. Cole. (2008). Oscillatory Lunatic Fringe activity is crucial for segmentation of anterior but not posterior skeleton. Development, 135.899-908. 3. E.T. Shifley and S.E. Cole. (2007). The vertebrate segmentation clock and its role in skeletal birth defects. Birth Defects Part C, 81.121-33. FIELDS OF STUDY Major Field: Molecular Genetics vii TABLE OF CONTENTS Page Abstract……………………………………………………………………………... ii Dedication………………………………………………………………………...... iv Acknowledgements………………………………………………………………… v Vita…………………………………………………………………………………. vii List of Tables……………………………………………………………………….. xii List of Figures……………………………………………………………………… xiii List of Abbreviations……………………………………………………………….. xvi Chapters: 1. Introduction…………………………………………………………………... 1 1.1 Introduction…………………………………………………………….… 1 1.2 Somitogenesis………………………………………………………….… 2 1.3 The Segmentation Clock…………………………………………………. 5 1.3.1 Cycling genes……………………………………………………… 6 1.3.2 Segmentation Clock Variations…………………………………… 9 1.3.3 Recent Clock Models……………………………………………… 10 1.4 The Wavefront…………………………………………………………… 12 1.5 The Notch Signaling Pathway…………………………………………… 13 1.5.1 Notch signaling plays multiple roles during somitogenesis………. 15 1.5.2 Notch pathway knockouts…………………………………………. 16 1.6 Human Disease…………………………………………………………... 18 1.6.1 Spondylocostal Dysostosis………………………………………... 18 1.7 The regulation of Lfng during somitogenesis……………………………. 23 viii 2. Lunatic Fringe is important for segmentation clock function during primary not secondary body formation………...……………………………………... 32 2.1 Introduction………………………………………………………………. 32 2.2 Materials and Methods………………………………………………….... 34 2.2.1 Targeted deletion of FCE1………………………………………… 34 2.2.2 Genotyping……………………………………………………….... 34 2.2.3 Whole mount in situ hybridization……………………………….... 35 2.2.4 Skeletal preparations………………………………………………. 36 2.2.5 Whole mount immunohistochemistry……………………………... 36 2.3 Results……………………………………………………………………. 36 2.3.1 Deletion of FCE1 from the Lfng locus perturbs clock-linked Lfng RNA expression……………………………………………………….. 36 2.3.2 The loss of Lfng expression in the segmentation clock perturbs normal skeletal development…………………………………………….. 37 2.3.3 Rostral-caudal somite patterning is partially rescued in Lfng∆FCE1/∆FCE1 embryos………………………………………………….. 39 2.3.4 Somites differentiate properly in Lfng∆FCE1/∆FCE1 embryos………... 41 2.3.5 The loss of cyclic Lfng expression in the posterior PSM perturbs oscillatory NOTCH1 activity……………………………………………. 41 2.3.6 Expression of oscillatory genes is differentially affected during primary and secondary body formation in Lfng∆FCE1/∆FCE1 embryos…….. 42 2.3.7 Wnt targets continue to cycle, and Brachyury expression is not disrupted in Lfng∆FCE1/∆FCE1 embryos……………………………………. 44 2.4 Discussion………………………………………………………………... 44 2.4.1 The FCE1 enhancer is necessary for cyclic expression of Lfng in Region I of the PSM………...…………………………………………... 44 2.4.2 Oscillatory Lfng expression and Notch signaling are critical for the proper segmentation during primary, but not secondary, body formation………………………………………………………………… 45 2.4.3 Lfng plays separable roles in the segmentation clock and R/C patterning………………………………………………………………... 47 2.4.4 Differential segmentation clock regulation at distinct levels of the axial skeleton?…………………………………………………………... 48 2.4.5 R/C patterning of anterior somites may affect ongoing segmentation during secondary body formation…………………………
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