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BEACH-DISSERTATION-2016.Pdf Towards mammalian retinal regeneration: cell signaling in re-differentiating mouse Müller glia and transcriptional regulation of the axonal guidance receptor EphA5 By Krista Marie Beach B.S, O.D. DISSERTATION In partial satisfaction of the requirements for the degree of PhD in PHYSIOLOGICAL OPTICS Presented to the Graduate Faculty of the College of Optometry University of Houston May, 2016 Approved: Dr. Deborah C. Otteson, PhD (Chair) Dr. Laura J. Frishman, PhD Dr. Donald A. Fox, PhD Dr. Steven W. Wang, PhD Committee in Charge DEDICATION I dedicate this dissertation to my parents, Col. Thomas N. Beach, M.D., and Jean M. Beach, B.A., and to my two sisters, Paula J. Beach, M.S., B.S. and Laura E. Beach, B.A., B.A. for their unconditional love and support during these years of incessant study and work. ii ACKNOWLEDGMENTS I wish to acknowledge my committee members for their guidance and support: Deborah C. Otteson, PhD – chair Laura J. Frishman, PhD Donald A. Fox, PhD Steven W. Wang, PhD I wish to acknowledge the past and present members of the Otteson lab, for their personal support, friendship, and occasional help with experiments: Amanda Gall, M.S Tia Petkova, O.D., PhD Tanya Geranpayeh, B.S Laila Pillai, B.S Mary Guirguis, B.S M. Joseph Phillips, PhD Steven Huynh, B.S Jian Bo Wang, PhD Micah Mesko, B.S Kuichen Zhu, PhD I wish to acknowledge also the following people: Librarians Suzanne Ferimer and Pamela Forbes of the Weston Petty UH Library for acquiring a great many hard-to-find articles and resources. Technology Trainer Chris Holthe of the MD Anderson UH Library for training in the use of several programs needed for this work. My family, my church, and my fellow graduate students, for their unconditional friendship and encouragement. iii DISSERTATION ABSTRACT Damage to the neural retina or optic nerve results in irreversible blindness because the mammalian retina cannot regenerate. For stem cell therapies to regenerate a functional retina, multiple challenges must be overcome. These include identification of stem cell sources, targeted differentiation into particular retinal neuronal cell types, delivery to the damaged area, survival and integration into the remaining retinal tissue, and establishment of appropriate connections between cells. Müller glia have been proposed as potential retinal stem cells, but attempts to re- differentiate Müller glia into retinal neurons have shown poor yield and incomplete differentiation. In cultured retinal progenitor cells, differential activation of MAPK versus STAT3 cell signaling pathways by the cytokine ciliary neurotrophic factor (CNTF) can promote neuronal versus glial differentiation, respectively. Specific Aim 1 measured MAPK and STAT3 signaling and neuronal versus glial gene expression in a mouse Müller cell line during re-differentiation and in response to treatment with CNTF. At different stages of in vitro re-differentiation, ImM10 Müller glia activated neurogenic MAPK and did not change gliogenic STAT3 signaling pathways. Exogenous CNTF did not alter MAPK or STAT3 signaling or the final expression levels of neuronal and glial genes. Overall, ImM10 cells do not respond to CNTF as previously reported for primary Müller glia, and their neurogenic potential is not altered by CNTF. Another challenge facing regenerative therapies will be re-establishment of appropriate connections between the retina and the visual areas of the brain. The axonal guidance receptors EphA5 and EphA6 are expressed in gradients across the retina, and changes in iv their expression alter the location of retinal ganglion cell (RGC) synaptic termination zones. Therefore, new RGCs will need to express EphA5 and EphA6 at levels appropriate for their retinal location. Specific Aim 2 addressed the regulatory hierarchy controlling EphA5 and EphA6 expression in the mouse retina. POU4F2 is a RGC-specific transcription factor that when deleted results in aberrant RGC projections, suggesting potential defects in expression of axonal guidance genes. KLF16 is a recently-identified regulator of EphA5 that is expressed after POU4F2 in newly-differentiated RGCs, but KLF16’s regulation and spatial expression patterns in the retina have not been characterized. In wild-type mice, KLF16 expression was equal in nasal retina comparted to temporal retina. In Pou4f2 knockout mice, expression of EphA5, EphA6, and Klf16 was unchanged compared to wild-type mice. Unexpectedly, there was increased expression of a commonly used reference gene, β-2 microglobulin (B2m), which has been implicated in retinogeniculate synaptic remodeling. Thus POU4F2 may repress B2m expression, but does not regulate EphA5, EphA6, or Klf16 expression. In conclusion, in vitro differentiation of ImM10 Müller glia results in conflicting activation of MAPK and STAT3 signaling that may, in part, explain the limited neurogenic potential of this cell line. This emphasizes the necessity of finding alternative stem cell sources for regenerative therapies. In the Pou4f2-/- mouse, axonal misrouting is not a result of dysregulation of EphA5, EphA6, or Klf16, but may involve dysregulation of B2m. Further studies are needed to identify the transcriptional regulators of axonal guidance genes that could be exploited to promote axon outgrowth, guidance, and connectivity of stem cell-generated RGCs for development of regenerative therapies. v TABLE OF CONTENTS DEDICATION ................................................................................................................ ii ACKNOWLEDGMENTS ............................................................................................. iii DISSERTATION ABSTRACT ..................................................................................... iv TABLE OF CONTENTS ............................................................................................... vi TABLE OF FIGURES ................................................................................................... ix TABLE OF TABLES ..................................................................................................... x Nomenclature ................................................................................................................. xi Dissertation introduction .....................................................................................1 RETINAL DEVELOPMENT ......................................................................................... 1 Retinal development overview ................................................................................... 1 Retinal ganglion cell axonal guidance and connectivity............................................. 9 RETINAL REGENERATION...................................................................................... 13 Stem cell sources....................................................................................................... 13 Cell signaling in a mouse Müller cell line during in vitro re-differentiation ....23 ABSTRACT .................................................................................................................. 23 INTRODUCTION ........................................................................................................ 25 Purpose ...................................................................................................................... 25 Müller glia as potential stem cells ............................................................................ 25 Ciliary neurotrophic factor (CNTF) and its receptor ................................................ 28 CNTF and receptor expression and localization ....................................................... 32 CNTF signaling pathways......................................................................................... 35 CNTF and MAPK signaling ..................................................................................... 37 CNTF and JAK/STAT signaling .............................................................................. 40 CNTF and PI3K signaling ........................................................................................ 46 CNTF signaling in the retina..................................................................................... 48 CNTF and CNTF receptor functions during development ....................................... 49 CNTF and the injury response .................................................................................. 53 METHODS ................................................................................................................... 64 Mice .......................................................................................................................... 64 Retinal dissociation cultures ..................................................................................... 64 ImM10 cell cultures .................................................................................................. 65 Reverse-transcriptase quantitative PCR (RT-qPCR) ................................................ 70 Western immunoblotting .......................................................................................... 71 Western immunoblot quantitative analysis ............................................................... 75 vi RESULTS ..................................................................................................................... 78 Neuronal versus glial gene expression in ImM10 differentiation cultures with CNTF ..................................................................................................................................
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