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Functional Analysis of Notch Signaling During Vertebrate Retinal Development

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Functional Analysis of Notch Signaling During Vertebrate Retinal Development Functional Analysis of Notch Signaling during Vertebrate Retinal Development The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Mizeracka, Karolina. 2012. Functional Analysis of Notch Signaling during Vertebrate Retinal Development. Doctoral dissertation, Harvard University. Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:10344746 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA © 2012 - Karolina Mizeracka All rights reserved. Advisor: Constance L. Cepko Karolina Mizeracka Functional analysis of Notch signaling during vertebrate retinal development Abstract The process of cell fate determination, which establishes the vastly diverse set of neural cell types found in the central nervous system, remains poorly understood. During retinal development, multipotent retinal progenitor cells generate seven major cell types, including photoreceptors, interneurons, and glia, in an ordered temporal sequence. The behavior of these progenitor cells is influenced by the Notch pathway, a widely utilized signal during embryogenesis which can regulate proliferation and cell fate decisions. To examine the underlying genetic changes that occur when Notch1 is removed from individual retinal cells, microarray analysis of single cells from wild type or Notch1 conditional knockout retinas was performed. Notch1 deficient cells downregulated progenitor and cell cycle marker genes, while robustly upregulating genes associated with rod genesis. Single wild type cells expressed markers of both rod photoreceptors and interneurons, suggesting that these cells were in a transitional state. In order to examine the role of Notch signaling in cell fate specification separate from its role in proliferation, Notch1 was genetically removed specifically from newly postmitotic cells. Notch1 deficient cells preferentially became cone photoreceptors at embryonic stages, and rod photoreceptors at postnatal stages. In both cases, this cell fate change occurred at the expense of the other cell types normally produced at that time. In addition, single cell profiling revealed that Inhibitor of differentiation 1 and 3 genes were robustly downregulated in Notch1 deficient cells. Ectopic expression of these genes during postnatal development in wild type retinas was sufficient to drive production of iii progenitor/Müller glial cells. Moreover, Id1 and 3 partially rescued the production of Müller glial cells and bipolar cells in the absence of Notch1, even in newly postmitotic cells. We propose that after cell cycle exit, retinal precursor cells transition through a period in which they express marker genes of several different cell types as they commit to a fate, likely endowed by their progenitor cell. Specifically, cells that will become bipolars or Müller glia depend on Id-mediated Notch signaling during this transitional state to take on their respective fates. iv This work is dedicated to my family: My grandparents, my parents, and Raphael v Acknowledgements I would like to thank all the members of the Cepko-Tabin and Dymecki labs for their scientific advice, emotional support, and constant supply of hilarity and fun during my graduate school years. Connie Cepko has been an amazing mentor for me. All of our interactions made it clear that she was greatly invested in my progress, partly because my project was a mix of her favorite things: retinal progenitor cells, Notch signaling, viruses, and single cell profiling, but also because she cared about my development as a scientist on a personal level. A deep-thinking, rigorous, and creative scientist, she is a rare example of a very successful, but grounded researcher. I would like to thank Cliff Tabin, the head of the Genetics department, for creating such an amazing environment for doing research. Also, I would like to thank Susan Dymecki for all her support over the years. I would like extend my gratitude to Tim Cherry and Jeff Trimarchi for being my friends and mentors when I first started in the lab. Also, I would like to thank all the wonderful Bay 3 members who made day-to-day life in the lab so enjoyable. These include: Laura Weiser, Christina Demaso, Didem Goz, and Patrick Allard. Patrick and I overlapped in the bay for a long time and spent many an evening talking about ridiculous things or loudly singing. I would like to thank all the members of the Cepko lab, and in particular: Nate Billings, Santiago Rompani, Mark Emerson, Takahiko Matsuda, Sui Wang, Wenjun Xiong, Natalia Surzenko, and Wenjia You. And of course, I have to acknowledge and vi thank the wonderful Ladies in the Tabin lab that are my very dear friends: Yana Kamberov, Jessica Lehoczky, Jenna Galloway, and Eddy McGlinn. These wonderful women have advised me both scientifically and personally, made the lab always an entertaining place, and are all fiercely talented scientists!! In addition, I would like to thank my amazing friends, Amanda Nottke (my twin!) and Jimmy Hu, for all their support and friendship. Tabin grad students, Tamar Katz, Amy Shyer, Alan Rodrigues, Aysu Ugur, and Matt Schwartz have made social life in the lab so much fun. I would like to thank my favorite classmates: Dan O’Connell, Joyce Tse O’Connell, Simon Trevino, Dave Shore, Mike Alpert, and Dana Christofferson. They have made my time in graduate school very memorable: full of delicious dinner parties, travel adventures, and ridiculous parties. Together, we went from the “G1 party group” to more “mature” adults. I will always cherish these amazing friends from grad school. I would like to thank my parents for their unending support. They have always put my education first, and have taught me directly, as well as by example, how to lead a fulfilling life. They had to overcome many adverse situations as immigrants in a new land, yet became successful because of their intelligence, good humor, discipline, and hard work. Their achievements serve as constant inspiration for my life. I also want to extend a big thank you to my wonderful grandparents, a team of superstars: Babcia Basia, Dziadek Gienek, Babcia Krysia, Dziadek Bolek. Despite having lived their lives in a difficult time and place, my grandparents are accomplished, generous, quick-witted, and warm people. They know a surprising amount about modern biology, keep close tabs on my life and work, and always make me feel incredibly supported. They are very special to me. vii I would also like to thank my amazing husband, Raphael Bruckner, for all his support over the years. Rafi has enriched my life in more ways than I can count or fully appreciate. He is the smartest person I know, kind, hard-working, fun, talented, and hilarious. As it turns out, you can meet your very best friend and life companion at a BBS retreat. viii Table of Contents Chapter 1. Introduction……………...…………………………………………….…….1 Chapter 2. Analysis of gene expression in wild type and Notch1 mutant retinal cells by single cell profiling………………………………………………..……………43 Chapter 3. Notch1 and downstream factors are required in newly postmitotic cells to inhibit the rod photoreceptor fate………………………………………………………..81 Chapter 4. Notch1 is required in embryonic newly postmitotic cells to inhibit the photoreceptor fate…………………………………………………………………..…..118 Chapter 5. Discussion………………………………………………………………….131 Appendix 1. Table of upregulated genes in Notch1 deficient cells…………..……......148 Appendix 2. Table of downregulated genes in Notch1 deficient cells………………...168 Appendix 3. Gene identities……………………………………………………………176 Appendix 4. Transcriptional role of cyclin D1 in development revealed by a genetic-proteomic screen…………………………………………………………..…180 ix Chapter 1 Introduction Retinal development and Notch signaling 1 The retina In examining the vertebrate eye, perhaps not too surprisingly, Charles Darwin found this organ “absurd in the highest possible degree” and deemed it impossible to have evolved through natural selection. To this day, the eye, and especially its neural component, the retina, continue to be fascinating structures that function more effectively than the most impressive manmade camera. The retina is a light sensitive tissue that lines the back of the eye. This highly specialized organ receives visual input from the outside environment, processes this information, and relays it to higher order visual processing centers in the brain. In understanding the function and development of the retina, we gain insight into how neural systems translate visual input into electrical impulses and how neural systems form during development. Although it still harbors mysteries, the retina is simpler in its neuroanatomy from the brain, thus making it an approachable part of the central nervous system in which to study neurogenesis. Over a hundred years ago, the neuroanatomist Santiago Ramon y Cajal and others, performed Golgi staining on retinal tissue, revealing its architecture, circuitry, and diverse set of neural cell types. The ultrastructure of the retina consists of three nuclear layers and two plexiform layers. The cell bodies of rod and cone photoreceptors are located in the outer nuclear layer (ONL). These cells protrude segments, which consist of both inner and outer components (OS and IS), into the subretinal space where they interact with the pigmented epithelium (RPE).
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