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Protocadherin-17 Function in Zebrafish Retina

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Protocadherin-17 Function in Zebrafish Retina PROTOCADHERIN-17 FUNCTION IN ZEBRAFISH RETINA DEVELOPMENT A Dissertation Presented to The Graduate Faculty of The University of Akron In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy Yun Chen December, 2012 PROTOCADHERIN-17 FUNCTION IN ZEBRAFISH RETINA DEVELOPMENT Yun Chen Dissertation Approved: Accepted: ______________________________ ______________________________ Advisor Department Chair Dr. Qin Liu Dr. Monte Turner ______________________________ ______________________________ Committee Member Dean of the College Dr. Richard Londraville Dr. Chand Midha ______________________________ ______________________________ Committee Member Dean of the Graduate School Dr. Brian Bagatto Dr. George R. Newkome ______________________________ ______________________________ Committee Member Date Dr. Bruce Cushing ______________________________ Committee Member Dr. Zhonghui Duan ii ABSTRACT Expression and function of protocadherin-17 (pcdh17) in zebrafish retinal development were analyzed in this study. Pcdh17 mRNA (pcdh17) expression pattern was characterized using whole mount in situ hybridization method. Antisense morphlino oligonucleotides (MOs) technique was used to determine pcdh17 function. Moreover, molecular mechanisms underlying pcdh17 expression and function were studied using proteomics. pcdh17 was expressed in developing zebrafish retina during critical stages of its development. Abnormal eye and retinal development was observed in developing zebrafish (49-72 hours post fertilization, hpf) injected with zebrafish pcdh17 specific MOs (pcdh17 morphants). The morphants had significantly smaller eyes and disrupted differentiated retinal cells (e.g. retinal ganglion cells and photoreceptors), due mainly to decreased cell proliferation as well as defects of nonneuronal cell differentiation. Proteomic analysis revealed that several hundred proteins were differentially expressed between wildtype embryos and pcdh17 morphants. A subset of spots showing the biggest differences were identified using Mass Spectrometry. These proteins included phosphoglycerate kinase, beta-actin-like protein, and glial fibrillary acidic protein (GFAP). Most of the identified proteins are involved in basic cellular metabolism and cellular structure, whereas GFAP is a molecule involved in Notch signaling pathway known to play a critical role in vertebrate retinal development. In addition, pcdh17 iii functions in retina development via regulating certain transcription factors, classical cadherins, homophilic reacting molecules and integrin-dependent adhesion. My research suggests that pcdh17 plays an important role in zebrafish retinal development, likely through involving multiple pathways including Notch-Delta, Wnt pathways. iv ACKNOWLEDGEMENTS I would never have been able to finish my dissertation without the guidance of my committee members, help from friends, and support from my family. I would like to express my deepest gratitude to my advisor, Dr. Qin Liu, for his excellent guidance, caring, patience, and providing me with an excellent atmosphere for doing research. I would like to thank Dr. Richard Londraville, who taught me the proteomics in past year. He also helped me to improve my scientific writing from the first year I was in US. It is a great technical support for my research. I would also like to thank Dr. Brian Bagatto and Dr. Bruce Cushing and Dr. Zhong-Hui Duan for guiding my research helping me to develop my background in physiology, neuroscience and computational biology. Also a thank to Dr. Jun Hu with his help in chemistry field. My colleagues as well as friends, Mark Dalman, Hope Ball, Donald Copeland, Sunil Bhattarai and Alicja Sochacka were always willing to help and give their best suggestions. My research would not have been possible without their help. I would also like to thank my family. They were always supporting me and encouraging me with their best wishes. They were always there cheering me up and stood by me through the good times and bad. My research is supported by grants from National Institutes of Health (R15 EY013879). v TABLE OF CONTENTS Page LIST OF TABLES ............................................................................................................ vii LIST OF FIGURES ......................................................................................................... viii CHAPTER I. INTRODUCTION ....................................................................................................... 1 II. LITERATURE REVIEW ............................................................................................ 4 III. PCDH17 mRNA EXPRESSION IN DEVELOPING ZEBRAFISH RETINA ......... 13 Materials and methods .......................................................................................... 13 Results ................................................................................................................... 16 IV. STUDY OF PCDH17 FUNCTION IN ZEBRAFISH RETINAL DEVELOPMENT 18 Materials and methods .......................................................................................... 18 Results ................................................................................................................... 24 V. MOLECULAR MECHANISM UNDERLYING pcdh17 FUNCTION .................... 39 Materials and methods .......................................................................................... 39 Results ................................................................................................................... 43 VI. DISCUSSION AND CONCLUSIONS ..................................................................... 57 Pcdh17 is essential in zebrafish eye development ................................................ 57 Loss-of-function techniques .................................................................................. 60 Possible mechanisms of pcdh17 function in retinal development ........................ 62 Summary and perspective ..................................................................................... 66 REFERENCES ................................................................................................................. 68 vi LIST OF TABLES Table Page 1. Effects of pcdh17 MOs injections on zebrafish development ...................................... 28 2. Effects of pcdh17 MOs injections on eye size .............................................................. 29 3. Effects of pcdh17 knockdown on zebrafish retinal development ................................. 32 4. Effects of vivo-pcdh17sMO injection on eye size ........................................................ 36 5. Effects of vivo-pcdh17sMO injection on zebrafish retinal development ..................... 38 6. Effects of pcdh17 knockdown on expression of transcription factors .......................... 45 7. Identification parameters for zebrafish MS analyzed protein bands ............................ 49 8. Gene ontology Biological Process Terms of the MS identified proteins ...................... 53 9. Gene ontology Molecular Function Terms of the MS identified proteins .................... 54 10. Gene ontology Cellular Component Terms of the MS identified proteins ................. 55 vii LIST OF FIGURES Figure Page 1. The dual function of β-catenin in cell adhesion and transcription .................................. 6 2. Expression of pcdhs from genomic DNA to functional protein ..................................... 8 3.Wide-type 72 hpf zebrafish eye. ...................................................................................... 9 4. pcdh17 expression in the developing zebrafish retina .................................................. 17 5. Gross morphological defects in pcdh17 morphants ...................................................... 25 6. Higher magnification of eye regions of embryos ......................................................... 25 7. Diagnostic RT-PCR demonstrating efficacy of the splice-blocking pcdh17 MO. ........ 26 8. Immunostaining analysis of retinal cell differentiation ................................................ 30 9. Retinal organization revealed by beta-catenin immunostaining.. ................................. 31 10. Apoptosis analysis using TUNEL staining ................................................................. 33 11. Histone-H3 immunostaining ....................................................................................... 34 12. Gross body and eye morphology in live control and vivo-morphant embryos........... 35 13. Immunostaining analysis of retinal cell differentiation in vivo-morphant retina ....... 37 14. Expression of transcription factors ............................................................................. 46 15. Blue stained gels of total proteins of developing zebrafish ........................................ 47 16. Coomassie blue staining 2-DE electrophoresis map. .................................................. 48 17. Immunohistochemstry confirmation of GFAP expression .......................................... 56 viii CHAPTER I INTRODUCTION Cadherins are cell adhesion molecules that play crucial roles in vertebrate development (Takeichi, 1990). During development of multicellular organisms, cadherins are involved in both cell-cell adhesion and cell signal transduction (Takeichi, 1988; Huelsken and Birchmeier, 2001; Yamaguchi, 2001). All cadherins share a similar structure: a large extracellular (EC) domain, a transmembrane domain and a cytoplasmic domain (Suzuki,
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