Graduate School ETD Form 9 (Revised 12/07) PURDUE UNIVERSITY GRADUATE SCHOOL Thesis/Dissertation Acceptance This is to certify that the thesis/dissertation prepared By Entitled For the degree of Is approved by the final examining committee: Chair To the best of my knowledge and as understood by the student in the Research Integrity and Copyright Disclaimer (Graduate School Form 20), this thesis/dissertation adheres to the provisions of Purdue University’s “Policy on Integrity in Research” and the use of copyrighted material. Approved by Major Professor(s): ____________________________________ ____________________________________ Approved by: Head of the Graduate Program Date Scott R. Hudson Meckelin 3 is Necessary for Photoreceptor Outer Segment Development Master of Science Teri L. Belecky-Adams Vince Gattone II Bonnie Blazer-Yost Teri L. Belecky-Adams Simon Atkinson 06/09/2011 Graduate School Form 20 (Revised 9/10) PURDUE UNIVERSITY GRADUATE SCHOOL Research Integrity and Copyright Disclaimer Title of Thesis/Dissertation: For the degree of Choose your degree I certify that in the preparation of this thesis, I have observed the provisions of Purdue University Executive Memorandum No. C-22, September 6, 1991, Policy on Integrity in Research.* Further, I certify that this work is free of plagiarism and all materials appearing in this thesis/dissertation have been properly quoted and attributed. I certify that all copyrighted material incorporated into this thesis/dissertation is in compliance with the United States’ copyright law and that I have received written permission from the copyright owners for my use of their work, which is beyond the scope of the law. I agree to indemnify and save harmless Purdue University from any and all claims that may be asserted or that may arise from any copyright violation. ______________________________________ Printed Name and Signature of Candidate ______________________________________ Date (month/day/year) *Located at http://www.purdue.edu/policies/pages/teach_res_outreach/c_22.html Meckelin 3 is Necessary for Photoreceptor Outer Segment Development Master of Science Scott R. Hudson 06/09/2011 MECKELIN 3 IS NECESSARY FOR PHOTORECEPTOR OUTER SEGMENT DEVELOPMENT A Thesis Submitted to the Faculty of Purdue University by Scott R. Hudson In Partial Fulfillment of the Requirements for the Degree of Master of Science August 2011 Purdue University Indianapolis, Indiana ii ACKNOWLEDGMENTS I would like to thank my committee members, Bonnie Blazer-Yost and Vince Gattone, for all their wisdom and guidance on my thesis project. To all the past and current lab members, your assistance, encouragement, and friendship throughout my graduate experience has been greatly appreciated. I would like to thank my advisor, Teri- Belecky-Adams, for allowing me the opportunity to continue towards my goal of becoming a scientist and for the invaluable graduate experience that has not only made me a better researcher, but a person as well. iii TABLE OF CONTENTS Page LIST OF TABLES v LIST OF FIGURES vi LIST OF ABBREVIATIONS viii ABSTRACT x CHAPTER 1. INTRODUCTION Eye Development 1 Mature Retinal Anatomy and Physiology 2 Vertebrate Photoreceptor Development 5 Outer Segment Development 7 Ciliopathies of the Retina 8 Meckel-Gruber Syndrome and Models of Meckel-Gruber 10 Meckelin and Related Proteins 11 CHAPTER 2. EXPERIMENTAL PROCEDURES WPK Rat Model 13 Molecular and Cellular Techniques Immunohistochemistry 14 TUNEL Labeling 15 H&E Staining 15 Transmission Electron Microscopy 16 Tissue Analysis and Statistics 16 CHAPTER 3. RESULTS Meckelin 3 is Found in the Developing and Mature Rat Retina 18 Histological Analysis of Retinae Isolated from Rat Mutant for Meckelin 3 19 Cell Loss in the WPK Mutant Retinae 20 Rudimentary Outer Segments are Initiated to Photoreceptor Degeneration in the WPK Mutant 23 iv Page CHAPTER 4. CONCLUSION Summary of Findings 24 Photoreceptor Outer Segment Development and Meckelin 3 25 Cell Death in Mks3 Mutants 25 Ciliopathies and Retinal Degenerations 27 Future Directions 29 LIST OF REFERENCES 32 v LIST OF TABLES Table Page 1.1 Ciliopathies with overlapping phenotypes 39 2.1 Primary Antibodies Used for Immunohistochemistry 40 vi LIST OF FIGURES Figure Page 1.1 Eye Formation During Embryonic Development 41 1.2 The Vertebrate Retina 42 1.3 Phototransduction in Photoreceptor Outer Segment 43 1.4 Light Pathway in the Retina 44 1.5 The Pathway of the Visual System 45 1.6 Structures of Photoreceptor Rods and Cones 46 1.7 Photoreceptor Connecting Cilium 47 1.8 Intraflagellar Transport in Photoreceptor Connecting Cilium 48 1.9 Outer Segment Formation by Invagination of Plasma Membrane 49 1.10 Outer Segment Formation by Pinocytosis 50 1.11 Development of Rod Photoreceptor 51 3.1 Meckelin 3 in the Developing Rat Retina 52 3.2 MKS3 is Found in Rods and Cones 53 3.3 MKS3 is Widely Expressed in the Rat Retina 54 3.4 Retinal Cell Layer Thickness and Photoreceptors in Mks3 Mutants 55 vii Figure Page 3.5 Cell-Type Specific Immunolabeling in the MKS3 Mutant Retina 56 3.6 Retinal Cell Counts 57 3.7 Cell Death of MKS3 Mutant Photoreceptors 58 3.8 Electron Micrscopy of Photoreceptor Outer Segments 59 viii LIST OF ABBREVIATIONS Autosomal Recessive Polycystic Kidney Disease ARPKD Bardet-Biedl Syndrome BBS Celsius C Centrosomal Protein CEP Embryonic day 9 E9 Frizzled FZD Ganglion Cell Layer GCL Glial Fibrillary Acidic Protein GFAP Hematoxylin and Eosin H&E Intraflagellar Transport IFT Inner Nuclear Layer INL Joubert Syndrome JBTS Kilograms kg Liter L Meckel-Gruber Syndrome MKS Microliter ul Microgram ug Microliter um [...]... positive for Sox2 (Figure 3. 3I-L) parvalbumin (Figure 3. 3M-P) and Brn3a (Figure 3. 3Q-T) Histological Analysis of Retinae Isolated from Rat Mutant for Meckelin 3 To further test the hypothesis that MKS3 is critical for the formation of photoreceptor outer segments, we utilized a rat with a spontaneous autosomal recessive mutation of the Mks3 gene, referred to as the rat Wistar polycystic kidney disease... the plasma membrane to form discs (Figure 1.9) 8 The second theory proposes that disc formation is due to pinocytosis of smaller vesicles that then fuse together to form a full disc that connects at the base of the outer segment, compressed by unknown mechanisms and then stacked together (Figure 1.10) [37 ] Disc formation is not limited to initial outer segment development, as it is continued all the... an outer segment process beyond a connecting cilium and xi rudimentary outer segment We conclude that MKS3 is not important for formation of connecting cilium and rudimentary outer segments (the inner stripe), but is critical for the development of mature outer segment processes The meckelin mutants showed similarities to human patients suffering from Leber’s congenital amaurosis We propose this may... were just beginning to form outer segments accompanied by laminated discs (Figure 3. 7A inset) However, the WPK mutant photoreceptor cilia form bulbus terminae without the formation of discs (Figure 3. 7B with inset) By P21, the WT retinae have completed outer segment formation with numerous laminated discs (Figure 3. 7C with inset) However, in the retinae of mutant rats lack outer segments with the cilia... 2011 Meckelin 3 is Necessary for Photoreceptor Outer Segment Development Major Professor: Teri L Belecky-Adams Ciliopathies with multiorgan pathology include renal cysts and eye pathology Retinal photoreceptors have connecting cilia joining the inner and outer segment that are responsible for transport of molecules to develop and maintain the outer segment process The present study evaluated meckelin. .. cilia in photoreceptor cell types that are involved in the outer segment development and maintenance Proper outer segment formation is crucial for photoreceptors to participate in phototransduction in order to allow for vision Eye diseases that are caused by irregular primary cilia in photoreceptors are known as retinal ciliopathies Retinal ciliopathies include several photoreceptor degenerative diseases... outer segment development and/or maintenance Outer Segment Development Photoreceptor cell genesis has been well studied; however, the formation of outer segment discs is still not well understood The first proposed theory suggests that the plasma membrane invaginates to form disc-like structures [36 ] As the connecting cilium extends, the plasma membrane forms a balloon-like structure around the connecting... through adulthood (Figure 1.11 [36 ]) Each day, the photoreceptor will shed 1/10th of its total discs on the distal most tip and that will get phagocytized by the retinal pigmented epithelium With either theory, the newly formed discs receive visual pigment that was made in the inner segment and transported to the outer segment through IFT Photoreceptors with mature outer segments can now take part in... inner to outer segment and the motor protein dynein to move components back to the cell body [35 ] While much information has been accumulated about the photoreceptors, there still remain many questions about the mechanisms of outer segment formation, protein transport through the connecting cilium, and the implications of alterations in protein trafficking to diseases affecting outer segment development. .. In this study, the expression patterns of meckelin have been studied using immunohistochemistry in the developing and mature rat retina Using the Wistar-Wpk 12 rat with a spontaneous mutation in the rMks3 gene [57], we previously showed that the formation of the photoreceptor outer segment development was dramatically impaired leading to loss of the photoreceptors [58] Herein, we found that the photoreceptors . UNIVERSITY GRADUATE SCHOOL Thesis/ Dissertation Acceptance This is to certify that the thesis/ dissertation prepared By Entitled For the degree of Is approved by the final examining committee:. Integrity and Copyright Disclaimer Title of Thesis/ Dissertation: For the degree of Choose your degree I certify that in the preparation of this thesis, I have observed the provisions of Purdue. all materials appearing in this thesis/ dissertation have been properly quoted and attributed. I certify that all copyrighted material incorporated into this thesis/ dissertation is in compliance