PHENOTYPIC AND MOLECULAR CHARACTERIZATION OF CATARACT IN THE TDRD7 KNOCKOUT MOUSE MUTANT by Carrie E. Barnum A thesis submitted to the Faculty of the University of Delaware in partial fulfillment of the requirements for the degree of Master of Science in Biological Sciences Spring 2015 © 2015 Carrie E. Barnum All Rights Reserved ProQuest Number: 1596827 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. ProQuest 1596827 Published by ProQuest LLC (2015). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, MI 48106 - 1346 PHENOTYPIC AND MOLECULAR CHARACTERIZATION OF CATARACT IN THE TDRD7 KNOCKOUT MOUSE MUTANT by Carrie E. Barnum Approved: __________________________________________________________ Salil A. Lachke, Ph.D. Professor in charge of thesis on behalf of the Advisory Committee Approved: __________________________________________________________ Robin W. Morgan, Ph.D. Chair of the Department of Biological Sciences Approved: __________________________________________________________ George H. Watson, Ph.D. Dean of the College of Arts and Sciences Approved: __________________________________________________________ James G. Richards, Ph.D. Vice Provost for Graduate and Professional Education ACKNOWLEDGMENTS I would like to thank my advisor Dr. Salil Lachke, he has been patient, helpful, understanding, critical when needed, and has always been there for me whether I knew it or not. He is a true friend as well as a true mentor and I will never forget that or stop appreciating it. I am so lucky that I was able to work with him. I know I am a better scientist because of it and he has fostered my appreciation of this field. I would like to thank my committee members, Dr. Melinda Duncan and Dr. Shawn Polson, I am sure they thought I would never graduate but they have been there for me with ideas and help throughout this process. I would like to especially thank Dr. Duncan for all her intellectual input and guidance in my times of need. I would like to thank my lab members, past and present, especially, Archana and Soma, they are the backbone of our lab and they have helped me keep focused and balanced, even when times are rocky. I would like to thank my family; they have been unwavering in their support and never stopped believing in me. I would like to thank my friends, especially Katie and Megan (I love you both). I would like to thank my dogs Pork and Beans, they are little lights that always shine in my life. Lastly, I would like to thank all of the animals used for this thesis. iii TABLE OF CONTENTS LIST OF TABLES ....................................................................................................... vii LIST OF FIGURES ..................................................................................................... viii ABSTRACT ................................................................................................................. xv Chapter 1 INTRODUCTION .............................................................................................. 1 1.1 Development of the Ocular Lens ............................................................... 1 1.2 Cataract ...................................................................................................... 5 1.3 Cell Types in the Lens ............................................................................. 11 1.4 Cellular Biology of the Ocular Lens ....................................................... 13 1.5 Transcriptional Control of Lens Development ........................................ 16 1.6 Post-Transcriptional Control in the Lens ................................................ 22 1.6.1 Alternative Splicing in the Lens .................................................. 24 1.6.2 miRNA Function in the Lens ...................................................... 27 1.6.3 RNA Binding Proteins in the Lens .............................................. 31 1.7 Tudor Domain Proteins in Cell Differentiation ....................................... 35 1.8 iSyTE: integrated Systems Tool for Eye gene Discovery ........................ 38 1.9 Mutations in Tdrd7 Cause Juvenile Cataracts ......................................... 42 2 METHODS AND MATERIALS ..................................................................... 46 1.10 Animals .................................................................................................... 46 1.11 DNA Isolation for Mouse Genotyping .................................................... 46 1.12 Mouse Genotyping .................................................................................. 48 1.13 Isolation of Embryos ............................................................................... 49 1.14 Light Microscopy .................................................................................... 50 1.15 Histology ................................................................................................. 50 1.16 Scanning Electron Microscopy (SEM) .................................................... 52 1.17 Immunofluorescence Staining ................................................................. 52 1.9 Lens Dissections ...................................................................................... 53 1.10 RNA Isolation Total RNA Isolation ........................................................ 54 1.11 Small and Large RNA Isolation .............................................................. 54 1.12 cDNA Synthesis ...................................................................................... 55 iv 1.13 Quantitative Reverse Transcription-PCR (qRT-PCR) ............................ 55 1.14 RNA Sequencing ..................................................................................... 56 1.15 Bioinformatics Analysis of RNA-Seq Data ............................................ 56 1.16 Phalloidin Whole Mount Staining of Lens .............................................. 58 1.17 Wheat Germ Agglutinin Staining of the Lens ......................................... 58 1.18 Protein-Protein Interaction Mapping Combined with iSyTE Expression ............................................................................. 59 1.19 RNA Immunoprecipitation ...................................................................... 60 1.20 Two-Dimensional Gel Electrophoresis and Mass Spectrometry ............. 63 1.20.1 2-D Gel Electrophoresis (2-DE) .................................................. 63 1.20.2 Protein and Peptide Identifications .............................................. 63 3 PHENOTYPIC CHARACTERIZATION OF LENS DEFECTS IN TDRD7 KNOCK OUT MOUSE MUTANTS ............................................................... 66 1.21 Light Microscopic Analysis of Eye Phenotype in Tdrd7 Mutants .......... 69 1.22 Histological Characterization of the Lens Phenotype in Tdrd7 Mutants .......................................................... 72 1.23 Analysis of the Tdrd7-/- Lens Defects by Scanning Electron Microscopy ................................................................ 74 1.24 Discussion ................................................................................................ 78 4 MOLECULAR CHARACTERIZATION OF TDRD7 KNOCKOUT MOUSE MUTANT LENS ............................................................................... 80 1.25 Investigation of Differentially Regulated RNAs in Tdrd7-/- Mouse Mutant Lenses ............................................................... 80 1.26 Tdrd7 2D DIGE ....................................................................................... 89 1.27 Discussion ................................................................................................ 93 5 CELLULAR CHARACTERIZATION OF FIBER CELL DEFECTS OF TDRD7 NULL MOUSE MUTANTS ............................................................... 98 1.28 Tdrd7 Protein-Protein Interaction Mapping ............................................ 99 1.29 RNA Immunoprecipitation (RIP) .......................................................... 103 1.30 Phallodin Staining of F-Actin ................................................................ 105 1.31 Wheat Germ Agglutinin Staining .......................................................... 109 1.32 Discussion .............................................................................................. 111 6 CONCLUSIONS AND FUTURE DIRECTIONS ......................................... 114 REFERENCES ..................................................................................................... 122 v Appendix A CURATED GENE LIST FROM TDRD7-/- MOUSE MUTANTS ............... 152 B COMPARISION OF RPKM AND COUNTS PER MILLION FOR CURATED GENE LIST ................................................................................ 154 C IACUC APPROVAL ...................................................................................... 156 vi LIST OF TABLES Table 1. Gene ontology from DAVID analysis. The 57 total genes above 1.5 fold change and expressed within the lens were group according to DAVID gene ontologies. This table corresponds