CHONDROITIN SULFATE AND KERATAN SULFATE PROTEOGLYCANS IN RETINAL AXON GROWTH AND GUIDANCE A THESIS SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL OF THE UNIVERSITY OF MINNESOTA BY Brian Douglas McAdams IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Steven C. McLoon, Advisor August 2018 © 2018 Brian Douglas McAdams Acknowledgements This was only possible due to the generous support and encouragement of my mentor, Dr. William Kennedy. He led the charge to make sure that this thesis would be completed. My new (and old) committee members have also been very encouraging, and I am deeply grateful. I would also like to thank Jerry Sedgwick for his assistance with his confocal microscopes in BIPL and for producing hypersensitized film for our lab. Also, I must thank the late Dr. Daniel Nordquist for his instruction in multiple photographic and laboratory techniques. I miss his keen insights and thoughtful suggestions (as well as the exasperated eye rolls). Thanks also to David Waid, Al Ernst and all the other McLoon lab members that made my time in that lab memorable. Thanks also to David Redish, Virginia Seybold, Virginia Goettl, Tim Gomez, Cheryl Stucky from Neuroscience, and Gwen, Mona, Ioanna, Rose, Patrick, Steve, Kenji, Shawn and Adam from the Kennedy lab for their encouragement and support. Thank you also to CC, JM, KS, BB and JT for your critical input into the process that made this possible. Thank you also to Willi Halfter for his support and encouragement and for the offer of the post-doc opportunity. Tracey, Lillie and my parents also were endlessly encouraging, and tirelessly supportive throughout my time in graduate school and in the years when I was working before the final push. This thesis could not have been completed without you. Wiley-Liss Publishers have kindly granted permission for use of my published article: McAdams, BD and McLoon, SC. The Role of Chondroitin Sulfate and Keratan Sulfate Proteoglycans in the Pathfinding of Retinal Axon Growth Cones in the Chick. Journal of Comparative Neurology. 352: 594-606, 1995 which is presented as chapter 2 in this thesis. I would also like to thank Dr. Richard Mayne (University of Alabama, Birmingham) for the gift of the 2C2 and 4D6 antibodies, Doug Fields (NIH) for his advice regarding the use of Campenot chambers, Ted Oegema for the bovine nasal proteoglycan, and Miriam Domowicz and Nancy Schwartz (University of Chicago) for the purified chick HNK1+ CSPG and B-aggrecan samples and multiple useful suggestions. Also, I would like to thank Lisa Nowak (University of Conn., i Storrs) for providing nanomelic chick eggs for the B-aggrecan study. This research was primarily supported by NIH grant EY05371 and the Graduate Program in Neuroscience. ii Dedication My family and friends were endlessly patient, understanding and helpful during my years in graduate school. They have suffered in their own ways by my side, literally or figuratively. I dedicate this thesis to them. Also, this thesis is dedicated to the powerless, to the voiceless, and to the visually impaired, and all those who selflessly and tirelessly work on their behalf. Finally, to my father, who taught me to ask the important questions, and to my recently deceased mother, who taught me how to find the answers. iii CHONDROITIN SULFATE AND KERATAN SULFATE PROTEOGLYCANS IN RETINAL AXON GROWTH AND GUIDANCE Abstract Axons of retinal ganglion cells grow from the eye to the visual centers of the brain during development. Evidence suggests that adhesive and anti-adhesive interactions between growing axons and the cells and extracellular matrix in their environment guide retinal axons to their central targets. Proteoglycans are a family of extracellular glycoproteins that could contribute to these interactions. The present study investigated the potential interaction and influence of glycosaminoglycans with retinal axons in situ and in vitro. Immunostaining showed both chondroitin sulfate and keratan sulfate proteoglycans throughout the retinotectal pathway during the period of axon growth. Retinal neurites extending from explants in culture were immunopositive for chondroitin sulfate indicating that retinal axons may contribute proteoglycans to the pathway. Retinal neurite behavior was examined in the presence of soluble glycosaminoglycans. High concentrations of chondroitin sulfate promoted retinal neurite growth on normally less adhesive substrates, which suggests that this glycosaminoglycan may promote neurite outgrowth in some conditions. Retinal axon growth was also examined in nanomelic chicks, mutants which do not secrete a large chondroitin sulfate proteoglycan, aggrecan. Aggrecan immunostaining was colocalized with retinal axons in normal embryos. For the parameters studied, retinal axon growth and guidance appeared unaffected in nanomelic mutants, which suggested that aggrecan was not essential for retinal axon growth or guidance. Its spatiotemporal distribution, however, suggests that aggrecan has other developmental roles in this system. Collectively, the data are ambiguous regarding the role of chondroitin sulfate and keratan sulfate proteoglycans relative to retinal axon growth during development. Proteoglycans are capable of influencing retinal axon growth in vitro, but whether they influence growth in vivo and the nature of this influence will require future investigations. iv Table of Contents Acknowledgements………………………………………………………………………….…………....i Dedication………………………………………………………………………………….….................iii Abstract…………………………………………………………………………………….………….......iv Table of Contents………………………………………………………………………….……………...v List of Figures……………………………………………………………………………….……….…...vi List of Tables……………………………………………………………………………………………...ix Chapter 1 - Introduction I. The Retinotectal Pathway of Chick………………………………………………………….1 II. Development of the Retinotectal Projection…………………………………...…….…....3 III. Mechanisms of Retinal Axon Motility and Guidance………………………….……..…..5 III. Molecules of the Retinotectal Pathway……………………………………..………....…20 IV. Proteoglycans and Retinotectal Development…………………….………..…….….....51 Chapter 2 Expression of Chondroitin Sulfate and Keratan Sulfate Proteoglycans in the Path of Growing Retinal Axons in the Developing Chick………………………………………………………..………67 Chapter 3 Effects of Exogenous and Native Glycosaminoglycans on Retinal Neurite Growth………..…....92 Chapter 4 Chick Brain Aggrecan Expression is SpatiotemporaIIy Regulated in the Retinotectal Pathway……………………………………………………………………………………………....…126 Chapter 5 Summary of Investigations into Potential Role of Chondroitin Sulfate and Keratan Sulfate Proteoglycans in the Development of the Path of Growing Retinal Axons in the Embryonic Chick …………………………………………………………………………………………………....155 Bibliography…………………………………………………………………………………….……....164 Appendix……………………………………………….………………………………………………..206 v List of Figures Chapter 1 Fig. 1. Illustration of a representative example of a proteoglycan …………………..………….....52 Chapter 2 Fig. 1. Micrographs of E2.5 retina and optic stalk with immunoreactivity to 3B3 and RA4……...83 Fig. 2. Micrographs of E5 retina showing immunoreactivity CS-56 and RA4……………...……..84 Fig. 3. Micrographs of E13 retina and optic nerve head showing immunoreactivity for CS, KS and RA4…………………………………………………………………….…......85 Fig. 4. Micrographs of E6 optic chiasm immunoreactivity with C6S, KS and RA4…………..…..86 Fig. 5. Micrograph of E10 optic tract shows immunoreactivity with C6S………………………….87 Fig. 6. Micrographs of the superficial tectum of E10 immunoreactivity with C6S, KS and RA4..88 Fig. 7. Micrographs of E 10 tectum immunoreactivity with C6S, KS and RA4 from a bienucleated embryo………………………………………………………………….……....89 Fig. 8. Micrographs of neurites from retinal explants immunoreactive to chondroitin sulfate…..90 Chapter 3 Fig. 1. Retinal explants grown on laminin (1g/ml, or 10g/ml) at three concentrations of C6S glycosaminoglycans……………………………………………………………..….117 Fig. 2. Neurite growth from retinal explants in C6S-supplemented media…………….………...118 Fig. 3. Retinal neurites growth on intact vitreous consisting of native chick chondroitin sulfate, hyaluronic acid and multiple collagens…………………………………………………….120 Fig. 4. Confocal micrograph of retinal neurites extending across cryosectioned vitreous immunostained for CS-56 and RA4 ……………………………………………..121 Fig. 5. Micrograph of retinal explant axon and fibroblast grown on a laminin and immunostained with CS-56 antibody to chondroitin sulfate…………………….………..122 Fig. 6. Preliminary data on the effects of exogenous glycosaminoglycans on retinal neurites...123 vi Fig. 7. Representative histogram of photometric gradient of fluorescence intensity for C4S glycosaminoglycan immunostaining in the optic tract ……………………………..……………...125 Chapter 4 Fig. 1. Micrographs of E3 retina and optic stalk immunostained for S103L and RA4…………..139 Fig. 2. Micrographs of E4 retina and optic stalk immunostained for CS, S103L and RA4……..140 Fig. 3. Micrographs of E5 optic nerve, chiasm and tract immunoreactive for CS, S103L and RA4………………………………………………………………………….141 Fig. 4. Micrographs of E7 retina are immunoreactive for CS, S103L and RA4………….…..….142 Fig. 5. Micrographs of E7 retina, optic nerves, chiasm and tracts with immunoreactivity for S103L and RA4………………………………………………………143 Fig. 6. Micrographs of E7 optic tectum immunostained for S103L and RA4……………….……144 Fig. 7. Micrographs E10 optic tectum immunostained for S103L and RA4………………….…..145 Fig. 8. Micrographs of E14 optic chiasm immunostained for S103L and GFAP……….………..146