UNIVERSITY OF CALIFORNIA Santa Barbara The Effect of Growth Differentiation Factor 6 on the Epithelial to Mesenchymal Transition in Retinal PiGmented Epithelium A dissertation submitted in partial satisfaction of the requirements for the deGree Doctor of Philosophy in Molecular, Cellular, and Developmental BioloGy By Amanda Mae Hurley Timm Committee in charGe: Professor Pete J. Coffey, Co-Chair Professor Dennis O. Clegg, Co-Chair Professor Anthony De Tomaso Professor Stuart Feinstein June 2019 The dissertation of Amanda Mae Hurley Timm is approved. Anthony De Tomaso Stuart Feinstein Dennis O. CleGG, Committee Co-Chair Pete J. Coffey, Committee Co-Chair May 2019 The Effect of Growth Differentiation Factor 6 on the Epithelial to Mesenchymal Transition in Retinal PiGmented Epithelium CopyriGht © 2019 By Amanda Mae Hurley Timm iii Acknowledgements First and foremost, I would like to thank the principle investiGator of my project, Dr. Pete J. Coffey. His encouragement, support, and Guidance made this work possible. HavinG a PI who believes in you and wants you to succeed makes Graduate school an easier place. I would also like to express my Gratitude for my co-advisor Dr. Dennis O. CleGG. His mentorship was invaluable durinG my time in Graduate school. I would also like to thank my committee members Dr. Stuart Feinstein and Dr. Anthony De Tomaso for their Guidance and insiGht on my project. I express my deepest appreciation to Dr. Monte J. Radeke. His patience, supervision, and assistance transformed me from an inquisitive student into the scientist I am today. I would also like to thank Carolyn M. Radeke for all of her technical expertise and support throuGhout the years. Dr. Rachael WarrinGton and Lindsay Bailey-Steinitz made cominG into lab a fun, enjoyable experience and I thank them for all of their knowledGe. My friends and family have been a huGe support system over the years and have been pillars of strenGth and encouragement. Finally, I would like to thank my husband, Robert, for all of his assistance. He supported me throuGhout this entire process and believed in me even when I did not believe in myself. This is as much yours as it is mine. iv VITA of Amanda Mae Hurley Timm May 2019 EDUCATION Ph.D. University of California, Santa Barbara 2019 Major: Molecular, Cellular, and Developmental Biology M.A. University of California, Santa Barbara 2017 Major: Molecular, Cellular, and Developmental BioloGy Certification in Management Practice University of California, Santa Barbara 2017 Major: TechnoloGy Management B.S. University of California, Davis 2010 Major: Biochemistry and Molecular BioloGy RESEARCH EXPERIENCE University of California, Santa Barbara Graduate Research Assistant 2013- Present The Effect of Growth Differentiation Factor 6 on the Epithelial to Mesenchymal Transition in Retinal PiGmented Epithelium. Dr. Pete J. Coffey, [email protected] Cedars-Sinai Medical Center Research Associate II 2010-2013 Characterization of neural Stem cell Growth and differentiation. Therapeutic use of GDNF-secretinG neural proGenitors in Amyotrophic Lateral Sclerosis. Dr. Clive N. Svendsen; [email protected] PUBLICATIONS GowinG G, Shelley B, StagGenborG K, Hurley A, Avalos P, Victoroff J, Latter J, Garcia L, Svendsen CN. Glial cell line-derived neurotrophic factor-secretinG human neural proGenitors show long-term survival, maturation into astrocytes, and no tumor formation followinG transplantation into the spinal cord of immunocompromised rats. Neuroreport. 2014; 25(6):367-72. Ebert A, Shelley B*, Hurley A*, Onorati M*, CastiGlioni V*, Patitucci T, Svendsen S, Mattis V, McGivern J, Schwab A, Sareen D, Kim HW, Cattaneo E, Svendsen C. EZ Spheres: A stable and expandable culture system for the Generation of pre-rosette multipotent stem cells from human ESCs and iPSCs. Journal of Stem Cell Research. 2013;10(3):417-27. *Contributed equally v ABSTRACTS Hurley A, Radeke M, Coffey P (April 2018). Growth Differentiation Factor 6 (GDF6) promotes irreversible epithelial to mesenchymal transition in retinal piGmented epithelial cells. Talk Given at the Neuroscience Research Symposium, UCSB, Santa Barbara, California Hurley A, Radeke M, Coffey P (October 2017). Growth Differentiation Factor 6 (GDF6) plays an inteGral role in irreversible epithelial to mesenchymal transition in retinal piGmented epithelial cells. Poster presented at the Southern California Biomedical Sciences Graduate Student Symposium, Cedars-Sinai Medical Center, Los AnGeles, California. Hurley A, Radeke M, Coffey P (April 2017). Growth Differentiation Factor 6 (GDF6) plays an inteGral role in irreversible epithelial to mesenchymal transition in retinal piGmented epithelial cells. Talk Given at the BioloGy of ReGenerative Medicine Conference, Wellcome Center in Hinxton, EnGland. Hurley A, Radeke M, Coffey P (May 2016). Growth Differentiation Factor 6 (GDF6) is a novel inducer of the epithelial-to-mesenchymal transition in retinal piGmented epithelial cells. Poster presented at ARVO, Seattle, WashinGton. Hurley A, Shelley B, CastiGlioni V, Onorati M, Svendsen S, Patitucci T, Mattis V, Sareen D, Kim HW, Heins B, McGivern J, Schwab A, Cattaneo E, Ebert A, Svendsen C (October 2012). EZ spheres: A characterization of a stable and expandable culture system for neural stem cells derived from hESC and hiPSC cultures. Poster presented at the Sociey for Neuroscience, New Orleans, Louisiana. AWARDS & FELLOWSHIPS The Jean Devlin Award in PharmacoloGy The GeorGe & Joy Rathmann Fellowship The Amgen Fellowship The Charles A. Storke II Fellowship TEACHING EXPERIENCE University of California, Santa Barbara TeachinG Assistant 2015 Course: Molecular Genetics of Prokaryotes University of California, Santa Barbara TeachinG Assistant 2013 Course: Introductory BioloGy I - Laboratory vi ABSTRACT The Effect of Growth Differentiation Factor 6 on the Epithelial-to-Mesenchymal Transition in Retinal Pigmented Epithelium By Amanda Mae Hurley Timm As retinal piGmented epithelium (RPE) cells are passaged, they underGo an irreversible epithelial-to-mesenchymal transition (EMT). We have shown previously that Growth differentiation factor 6 (GDF6), a member of the transforminG Growth factor-beta (TGFβ) family, is hiGhly upreGulated in RPE cells that have lost the capacity to obtain an epithelial phenotype. We hypothesize that GDF6 plays an inteGral role in the irreversible transition of an RPE cell from an epithelial state to a mesenchymal state. To test this hypothesis, we overexpressed GDF6 in differentiation competent RPE and assessed the effects on phenotype and Gene expression. To evaluate what receptors and siGnalinG pathways miGht mediate GDF6’s effects, cells were also treated with an Alk5 inhibitor (Repsox), an Alk2/3/6 inhibitor (LDN-193189), or both in combination. Passage 0 RPE transduced with GDF6 produce siGnificantly less piGmentation than cells infected with an empty vector control. This reduction in piGmentation is accompanied with a chanGe in cell morpholoGy; the control cells maintain a cuboidal morpholoGy and the GDF6 expressing cells have a spindle-like morpholoGy. Quantitative PCR analysis reveals that RPE cells transduced with GDF6 siGnificantly upreGulate known EMT markers like ACTA2, CTGF, and COL1A1 and downreGulate classical RPE markers such as LRAT and PMEL compared to control cells. Both RepSox and LDN-193189 have the ability to reverse the GDF6 phenotype. vii While the phenotype is recovered, RNA-seq analysis reveals GDF6-mediated reGulation of Genes that are unaffected by inhibitors, such as TGFβ1, MSX2, and CDH1. GDF6 is involved in the EMT process in RPE. We believe GDF6 upreGulates TGFβ1, which in turn promotes EMT. The ability to revert back to an epithelial cell is inhibited by GDF6. Cells exposed to GDF6 will prematurely underGo EMT, simultaneously downreGulatinG traditional RPE markers while upreGulatinG EMT and wound response markers. Inhibition of the BMP receptors rescues the GDF6 phenotype, therefore inhibition of GDF6 may be inteGral in prolonGinG the inteGrity and functional lifespan of the RPE. As such, inhibition of GDF6 may help restore RPE to their epithelial state in diseases which affect RPE such as proliferative vitreoretinopathy and age-related macular deGeneration. viii TABLE OF CONTENTS CHAPTER I: INTRODUCTION ........................................................................................................ 1 1.1: Retinal Pigmented Epithelium (RPE) ....................................................................... 1 1.1.1: Barrier Function ......................................................................................................... 1 1.1.2: Support ...................................................................................................................... 2 1.1.3: Visual Cycle ................................................................................................................ 3 1.1.4: Age-Related Defects .................................................................................................. 4 1.2: Epithelial-to-Mesenchymal Transition (EMT) .......................................................... 4 1.2.1: Type 1 EMT ................................................................................................................ 5 1.2.2: Type 2 EMT ................................................................................................................ 5 1.2.3: Type 3 EMT ...............................................................................................................