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First Line of Title β1-INTEGRIN MAY REGULATE EGR1 (EARLY GROWTH RESPONSE 1) WITHIN THE LENS by Anne M Terrell A thesis submitted to the Faculty of the University of Delaware in partial fulfillment of the requirements for the degree of Masters of Science in Biological Sciences Summer 2013 © 2013 Anne M Terrell All Rights Reserved β1-INTEGRIN MAY REGULATE EGR1 (EARLY GROWTH RESPONSE 1) WITHIN THE LENS by Anne M Terrell Approved: __________________________________________________________ Melinda K. Duncan, Ph.D. Professor in charge of thesis on behalf of the Advisory Committee Approved: __________________________________________________________ Randall L. Duncan, 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, Melinda K. Duncan, for all her support throughout this project. She has helped me to become a better scientist, writer and presenter. I would also like to thank all past and present Duncan lab members, with a special thanks to Abby Manthey, Yan Wang, and Yichen Wang. Their help and guidance throughout my time in the lab was incredible. Abby Manthey was an amazing mentor in the lab and collaborator on the RNA-Seq project. Without Yan Wang’s expertise in lens cataract surgeries, I would have never been able to finish the second half of my thesis. Thank you to Yichen Wang for helping with the Egr1 staining and continuing this amazing project. I’d like to thank Shawn Polson and John H. McDonald for their help on bioinformatics and statistics. Finally, I would like to thank my family and my amazing friends for their unconditional support and encouragement. I would like to give special thanks to my Mom, for always lending an ear, even when she had no idea what I was going on about. Thank you to Matt Smith for always finding a way to make me laugh. And my amazing graduate friends: Kaitlin Read (Jay Read), Deb Yannessa, Mollee Crampton, Rick Davis, Karl Franke, Stephanie Luff, Tom Carr, Wachen Peters, Casey Lumpkin, Amanda Fisher, Katie Robbins, Megan Carpenter… without your friendship and help, I don’t know how I would have made it through. iii TABLE OF CONTENTS LIST OF TABLES ........................................................................................................ vi LIST OF FIGURES ....................................................................................................... ix ABBREVIATIONS ...................................................................................................... xv ABSTRACT ............................................................................................................... xvii Chapter 1 INTRODUCTION .............................................................................................. 1 1.1. Lens Development ..................................................................................... 1 1.2 Cell Types in the Lens ............................................................................... 5 1.3 EMT ........................................................................................................... 7 1.4 Integrins ................................................................................................... 11 1.5 Integrin Adhesome .................................................................................. 17 1.6 β1-integrin: Required for Lens Epithelial Cell Maintenance and Survival ................................................................................................... 19 2 METHODS AND MATERIALS ..................................................................... 24 2.1 Animals .................................................................................................... 24 2.2 Cataract Surgeries .................................................................................... 25 2.3 DNA Isolation for Mouse Genotyping .................................................... 26 2.4 Genotyping .............................................................................................. 27 2.5 Isolation of embryos ................................................................................ 28 2.6 Immunofluorescence ............................................................................... 30 2.7 RNA Isolation .......................................................................................... 32 2.8 Simultaneous RNA/Protein Purification ................................................. 36 2.8.1 RNA Isolation .............................................................................. 36 2.8.2 Protein Purification ...................................................................... 37 2.9 cDNA Synthesis ...................................................................................... 38 2.10 quantitative Reverse-Transcription PCR (qRT-PCR) ............................. 39 2.11 RNA Sequencing ..................................................................................... 41 3 IDENTIFICATION OF DIFFERENTIALLY EXPRESSED GENES IN LENSES LACKING β1-INTEGRIN ............................................................... 46 iv 3.1 Global Transcriptome Analysis: Microarray vs RNA-sequencing ......... 46 3.2 Global analysis shows a subset of genes that may be responsible for the partial fibrotic β1-integrin phenotype ................................................ 48 3.3 Discussion ................................................................................................ 51 4 EGR1: PROPOSED INVOLVMENT IN THE LENS ..................................... 62 4.1 Egr1, Early Growth Response 1 .............................................................. 62 4.2 Nab2 negatively regulates Egr1 expression levels .................................. 65 4.3 Egr1 stimulation ...................................................................................... 66 4.4 Egr1 has the potential to mediate the β1-integrin cKO embryonic phenotype ................................................................................................ 68 4.5 Results ..................................................................................................... 71 4.6 Discussion ................................................................................................ 77 5 A POTENTIAL ROLE FOR EGR1 IN POSTERIOR CAPSULAR OPACIFICATION ........................................................................................... 91 5.1 Cataracts, Surgery and Outcomes ............................................................ 91 5.2 Time Course of the Mouse Cataract Surgery Model ............................... 93 5.3 The Role of Egr1 after Fiber Cell Removal ............................................ 94 5.4 Egr1: a possible regulator of early gene changes in PCO in wild type lenses ....................................................................................................... 98 5.5 Egr1 absence from the lens does not cause a decrease in the fibrotic response 5 days after surgery .................................................................. 99 5.6 Discussion .............................................................................................. 100 6 CONCLUSIONS AND FUTURE DIRECTIONS ......................................... 108 REFERENCES ........................................................................................................... 117 Appendix A COMPARISON OF LENSES IN DIFFERENT MOUSE STRAINS ............ 132 B IACUC LETTER OF APPROVAL ................................................................ 190 v LIST OF TABLES Table 2.1 Primers used for PCR analysis of mice. β1-integrin and Egr1 primers recommended by the Jackson Laboratory. MLR10 sequence obtained from Michael Robinson (Miami University of Ohio, Oxford, Ohio) (Zhao et al., 2004). .................................................................................. 28 Table 2.2 Antibodies used for immunofluorescence. Abbreviation, A:M – acetone:methanol. .................................................................................... 31 Table 2.3 Total RNA yield and quality of lens samples collected for analysis. The RNA yield (ng), calculated based on concentration and amount of RNA originally obtained (70 μl – embryo, 30 μl – PCO), is listed first, the quality of the RNA is listed in parenthesis. ....................................... 34 Table 2.4 Number of lenses collected for each biological replicate at each time point. These lenses were then used for extracting RNA. The number of wild type E15.5 lenses collected for RNA-Seq is higher than those isolated for the conditional knock out. This is because the wild type was collected first, and it was unclear how many would be needed for quality RNA. ........................................................................................... 35 Table 2.5 Primer sequences and citations used for qRT-PCR analysis. .................. 40 Table 3.1 Truncated table of differentially expressed genes in comparison between Sip1 floxed/floxed no-Cre (AG) and C57Bl/6 wild type strains. This comparison was done comparing Sip1 WT to C57 WT (Sip1WT expression/C57 WT expression), meaning that Snhg9 is expressed higher in the C57Bl/6 wild type. Only genes changed more than 2 fold shown. The full list of genes is listed in Appendix 1. ........... 54 vi Table
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