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Notchless Interacts with Multiple Signaling Pathways During Mouse Peri-Implantation Development Chiao-Ling Lo Purdue University

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Notchless Interacts with Multiple Signaling Pathways During Mouse Peri-Implantation Development Chiao-Ling Lo Purdue University Purdue University Purdue e-Pubs Open Access Dissertations Theses and Dissertations Fall 2013 Notchless Interacts with Multiple Signaling Pathways during Mouse Peri-Implantation Development Chiao-Ling Lo Purdue University Follow this and additional works at: https://docs.lib.purdue.edu/open_access_dissertations Part of the Animal Sciences Commons, and the Genetics Commons Recommended Citation Lo, Chiao-Ling, "Notchless Interacts with Multiple Signaling Pathways during Mouse Peri-Implantation Development" (2013). Open Access Dissertations. 105. https://docs.lib.purdue.edu/open_access_dissertations/105 This document has been made available through Purdue e-Pubs, a service of the Purdue University Libraries. Please contact [email protected] for additional information. Graduate School ETD Form 9 (Revised 12/07) PURDUE UNIVERSITY GRADUATE SCHOOL Thesis/Dissertation Acceptance This is to certify that the thesis/dissertation prepared By Chiao-Ling Lo Entitled Notchless Interacts with Multiple Signaling Pathways during Mouse Peri-Implantation Development Doctor of Philosophy For the degree of Is approved by the final examining committee: Amy C. Lossie Chair Ann L. Kirchmaier Shihuan Kuang Yuk Fai Leung To the best of my knowledge and as understood by the student in the Research Integrity and Copyright Disclaimer (Graduate School Form 20), this thesis/dissertation adheres to the provisions of Purdue University’s “Policy on Integrity in Research” and the use of copyrighted material. Approved by Major Professor(s): ____________________________________Amy C. Lossie ____________________________________ Approved by: Christine A. Hrycyna 11/20/2013 Head of the Graduate Program Date i NOTCHLESS INTERACTS WITH MULTIPLE SIGNALING PATHWAYS DURING MOUSE PERI-IMPLANTATION DEVELOPMENT A Dissertation Submitted to the Faculty of Purdue University by Chiao-Ling Lo In Partial Fulfillment of the Requirements for the Degree i of Doctor of Philosophy December 2013 Purdue University West Lafayette, Indiana ii ACKNOWLEDGEMENTS I would like to thank my major advisor Dr. Amy Lossie, for her mentoring and all the support, financially and emotionally, throughout the course of my PhD. I could not have completed my thesis without her. Thanks to my committee members, Dr. Ann Kirchmaier, Dr. Shihuan Kuang, and Dr. Yuk Fai Leung for their advice and critical review. I also want to thank Dr. William Muir for teaching me NGS data analysis, and Dr. Joseph Garner for his sophisticated statistical analysis on our projects. I appreciate the help from all my former and present labmates: Jon, Giovana, Katherine, Missy, Jeremy, Feichen, Xiao, Jennifer and Muyi. Moreover, I want to thank my parents, my brothers and all of my friends and colleagues in Taiwan and Purdue for their encouragement and care. They let me know that I am loved and I am not alone. Last but not least, I thank the mice that were sacrificed for this project; truly, without them, I could not have accomplished this research. ii iii TABLE OF CONTENTS Page LIST OF TABLES ................................................................................................. vi LIST OF FIGURES ............................................................................................... vii ABSTRACT ......................................................................................................... viii CHAPTER 1. INTRODUCTION ............................................................................. 1 1.1 Signaling During Peri-implantation Developmet………………………. ...... 1 1.2 NOTCH Signaling…………………………………………………………. ..... 5 1.3 Wnt Signaling…………………………………………………………….. ..... 10 1.4 Ribosomal Biogenesis……………………………………………………… . 11 1.5 Generation and Phenotype of l11Jus1 and l11Jus4……………………… 14 1.6 NOTCHLESS in the ribosomal biogenesis pathway……………………... 16 1.7 P53 Regulation……………………………………………………………….. 17 1.8 Objective……………………………………………………………………… 20 CHAPTER 2. IDENTIFICATION OF 129S6/SVEVTAC-SPECIFIC iii POLYMORPHISMS ON MOUSE CHROMOSOME 11 ....................................... 22 2.1 Introduction………………………..………………………..………………… 22 2.2 Methods………………………..………………………..…………………… . 25 2.3 Results…………………………………..…………………………………… . 28 2.4 Discussion………………………………………………………………… ..... 46 CHAPTER 3. ENU MUTAGENESIS REVEALS THAT NOTCHLESS HOMOLOG 1 (DROSOPHILA) AFFECTS CDKN1A AND SEVERAL MEMBERS OF THE WNT PATHWAY DURING MURINE PRE-IMPLANTATION DEVELOPMENT ... 50 3.1 Introduction………………………..………………………..………………… 50 3.2 Methods………………………..………………………..…………………… . 54 iv Page 3.2.1 Mouse Strains, Meiotic Mapping and Generation of Mutants ........... 54 3.2.2 Embryo Analysis ............................................................................... 55 3.2.3 Histology ........................................................................................... 56 3.2.4 Candidate Gene interrogation .......................................................... 56 3.2.5 Notch Pathway Expression of l11Jus1 mutants at E3.5 ................... 56 3.2.6 PCR Array Data Analysis for Gene Expression ............................... 60 3.2.7 Caspase 3 Detection ........................................................................ 60 3.2.8 Quantitative RT-PCR by Taqman ..................................................... 61 3.2.9 Statistical Methods ........................................................................... 62 3.3 Results………………………..………………………..……………………... 63 3.3.1 Phenotypic Analysis of l11Jus1 and l11Jus4 ................................... 63 3.3.2 Positional Cloning of l11Jus1 and l11Jus4 ....................................... 66 3.3.3 The Nle1 locus ................................................................................. 68 3.3.4 Expression Analysis of the Notch Pathway in Mutant Embryos ....... 70 3.3.5 Confirmation of Cdkn1a in Different Stages in Pre-Implantation ...... 73 3.3.6 Apoptosis Occurs at E4.5 in Nle1 Mutants ....................................... 74 3.3.7 qRT-PCR Analysis of Trp53 in Mutant Embryos .............................. 76 3.4 Discussion………………………..………………………..………………… . 77 CHAPTER 4. NLE1 MISSENSE MUTATIONS TIGGER TRP53 ACTIVATION iv AND DISRUPTION IN RIBOSOMAL BIOGENESIS DURING MURINE PRE- IMPLANTATION DEVELOPMENT ...................................................................... 81 4.1 Introduction………………………..………………………..………………… 81 4.2 Methods………………………..………………………..…………………… . 86 4.2.1 Immunofluorescence ........................................................................ 86 4.2.2 Quantitative RT-PCR ........................................................................ 87 4.2.3 Sample Preparation for rRNASeq .................................................... 88 4.2.4 Data analysis .................................................................................... 89 4.3 Results………………………..………………………..……………………... 91 4.3.1 TRP53 is Activated in Mutant Embryos ............................................ 91 v Page 4.3.2 Altered Gene Expression in Cell Cycle and Apoptosis Pathways .... 93 4.3.3 rRNA processing is disrupted in the Nle1 mutants ........................... 97 4.4 Discussion………………………..………………………..……………… .. 102 CHAPTER 5. DISCUSSION .............................................................................. 105 5.1 NLE1 and Its Potential Biological Function in Ribosomal Biogenesis .. 105 5.2 The rRNASeq study………………………..………………………………. 110 5.3 Mutations in NLE1 Affect Wnt Signaling…………………………………. 112 5.4 Altered Nucleolar Structure and p53 Activation in the Mutant………….114 5.5 Model for Signaling Regulation in Nle1 Mutants…………………………116 LIST OF REFERENCES ................................... ……….………. ………………. 119 VITA ……….………. ……………………………………….……….……………...141 v vi LIST OF TABLES Table ................................................................................................................ Page Table 1.1 Mutant lethality / phenotypes of the NOTCH signaling in mice ............. 9 Table 2.1 Primers used for this study .................................................................. 26 Table 2.2 Polymorphisms in the Nle1 locus ........................................................ 31 Table 2.3 Mettl16 polymorphisms ....................................................................... 34 Table 2.4 Psmd11 polymorphisms ...................................................................... 36 Table 2.5 Evi2a polymorphisms .......................................................................... 36 Table 2.6 Cct6b polymorphisms .......................................................................... 37 Table 2.7 Rffl polymorphisms………………………………………………………...39 Table 2.8 Ap2b1 polymorphisms……………………………..………………….…….41 Table 2.9 Summary of polymorphism analysis …………………………………….44 Table 3.1 Primers used for this study .................................................................. 59 Table 3.2 Time of death for Nle1 mutants ........................................................... 65 Table 3.3 X2 analysis of selected timed matings ................................................. 66 vi Table 3.4 Logistic multiple regression analysis of caspase 3 detection .............. 76 Table 4.1 Primers used for this study .................................................................. 87 Table 4.2 Logistic multiple regression analysis of active TRP53 detection ......... 93 Table 4.3 Average coverage of each rRNA subunits .......................................... 98 vii LIST OF FIGURES Figure .............................................................................................................
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