TRANSCRIPTIONAL CONTROL OF HYPOTHALAMIC TANYCYTE DEVELOPMENT By Ana Lucia Miranda-Angulo A dissertation submitted to the Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland March 2013 © 2013 Ana Lucia Miranda-Angulo All Rights Reserved Abstract The wall of the ventral third ventricle is composed of two distinct cell populations; tanycytes and ependymal cells. Tanycytes support several hypothalamic functions but little is known about the transcriptional network which regulates their development. We explored the developmental expression of multiple transcription factors by in situ hybridization and found that the retina and anterior neural fold homeobox transcription factor (Rax) was expressed in both ventricular progenitors of the hypothalamic primordium and differentiating tanycytes. Rax is known to participate in retina and hypothalamus development but nothing is known about its function in tanycytes. To explore the role of Rax in hypothalamic tanycyte development we generated Rax haploinsufficient mice. These mice appeared grossly normal, but careful examination revealed a thinning of the third ventricular wall and reduction of tanycyte and ependymal markers. These experiments show that Rax is required for tanycyte and ependymal cell progenitor proliferation and/or survival. Rax haploinsufficiency also resulted in ectopic presence of ependymal cells in the α2 tanycytic zone were few ependymal cells are normally found. Thus, the presence of ependymal cell in this zone suggests that Rax was required for α2 tanycyte differentiation. These changes in the ventricular wall were associated with reduced diffusion of Evans Blue tracer from the ventricle to the hypothalamic parenchyma. Furthermore, we have provided in vivo and in vitro evidence suggesting that RAX protein is secreted by tanycytes, and subsequently internalized by adjacent and distal cells. Finally, we generated an inducible Rax:CreERT2 mouse line which we have found to be selectively active in hypothalamic tanycytes, and will be a useful tool for manipulating gene function in these cells. In conclusion, we ii have provided evidence that during development Rax is necessary to control tanycyte and ependymal cell progenitor proliferation and for α2 tanycyte differentiation. We also show that RAX protein is secreted from tanycytes and internalized by proximal and distal cells. Finally, the inducible Rax:CreERT2/+ mouse line generated in this study will provide an invaluable tool for future studies of tanycyte development and function. Thesis advisor: Seth Blackshaw, Ph.D. Reader: Adam Kaplin M.D, Ph.D. iii Acknowledgements I would like to thank my advisor Seth Blackshaw for his guidance, understanding and support over the past seven years. Seth´s passion for science has always been an inspiration. His enthusiasm for discovering new things and for accomplishing complex projects always challenged me beyond what I thought I could do both personally and scientifically. He gave me enough freedom to think for myself, solve problems and generate new ideas while at the same time being supportive and providing needed guidance. I thank him for believing that I could always accomplish more. I also want to thank all the current and previous members of Seth´s lab who I had the opportunity to interact with. They made my days much better and the challenges easier. I especially want to thank Mardi Byerly, a previous postdoc in Seth´s lab, who helped me during some of the hardest times. She not only encouraged me but also spent many hours helping me with critical experiments even when she was exhausted. I want to thank Jimmy de Melo for his discussions about science and life, and for letting me use his critical eye and experience. He was encouraging and supportive at difficult times and did not hesitate to listen and help. I would like to thank Hong Wang for her dedication, efficiency, and patience when performing all my genotyping and lab ordering. Her assistance made many of my experiments possible. I would like to thank Thomas Pak for his dedication and efforts to make the functional characterization of our knock-in mice possible. I also would like to thank Lizhi, our previous technician and a good friend. She was always willing to make probes or anything else I needed. Her smile and willingness to work were inspiring. I thank Janny, a rotating undergrad, who was a great companion during the last months of my graduate training and who helped me with image analysis. I iv thank Thuzar and Liz for their gracious ways and ability to create a friendly lab environment. I also would like to thank them for giving me a very nice photo album that will never let me forget the people I met during this period of my life. Thanks to Juan for letting me speak Spanish to him in the lab to feel a little closer to my home country, and Kai for sharing his enthusiasm and curiosity for science which reminded me how much fun science can be. I want to thank Dan, who joined Seth’s lab at the same time than I did. Together we accepted the challenge of studying tanycytes for the first time in this lab, his findings and technical expertise were very helpful for the development of my experiments. Thanks to Erin, Eric, and E.J. for their positiveness and friendship at all times. I would also like to thank Nicole and Aki, the first two members of Seth’s lab, who welcomed me and guided me during my first years of training. I want to thank Vani, a former undergrad in the lab. She helped start our great adventure studying tanycytes in Seth’s lab. I want to thank all the people from other labs in the neuroscience department at Johns Hopkins who helped me with advice and/or reagents, especially Caitlin Engelhard, a previous member of David Ginty’s lab. Caitlin kindly and patiently walked me through the generation of the knock-in constructs. I would also like to thank Shin Kang, a previous member of Dwight Bergles lab. He gave me helpful advice and contributed reagents for the generation of the knock-in constructs. Many thanks to Michelle Pucak for her guidance with experiments and valuable help with the Imaris software and to Claudia Ruiz, a neuroscience graduate student who rotated in our lab and made possible to obtain data necessary to start the knock-in mouse project. v People from Hopkins facilities were indispensable to me during my graduate studies. I want to give a special thanks to Barbara, Mike, Loza, Scot and Ester from the Microscope Core Facility for their hospitality, friendliness, and willingness to assist me in every step of imaging and analysis. I also thank Chip and Holly from the Johns Hopkins transgenic core. They made possible the generation of the knock-in mice. Also, thanks to the people in the mouse facility who took great care of my mice. I want to specially thank the members of my thesis committee: David Ginty, Nicholas Gaiano, Hongjun Song, and Adam Kaplin; for their guidance not only in the academic area, but also during personal difficulties. They were all very helpful in pushing me to accomplish my goals. I want to thank Beth and Rita, as well as other administrative members who helped make possible the existence of this wonderful graduate program, and for making me feel as an important member of the neuroscience department family. I thank my previous advisors during my postdoctoral fellowship at NIMH. Dr. Cynthia Weickert and Daniel Weinberger were both great mentors and gave me a great deal of support and allowed me to be able to move toward my graduate training at Hopkins. I want to thank the PAHO and NIH for giving me the opportunity to obtain my postdoctoral training at NIMH and for their financial support. Also, I thank the Universidad de Antioquia in Colombia for its financial support during all my training in the United States. I especially thank Dr. Carlos Palacio, the vice dean of the Universidad de Antioquia, who helped me solve all kind administrative difficulties necessary for me to finish my graduate training. vi I thank all our collaborators who donated mice or helped with experiments: Tomomi Shimogori, Jin Woo Kim, Peter Mathers, Gordon Fishell, Xinzhong Dong, and Jeremy Nathans. I want to thank my friends Lindsay, Lauren, Prya, Rob, Heather and Mala who made my time at Hopkins and in Baltimore a happy one. Thanks to all who kindly helped editing this thesis Mardi, Jimmy, Lindsay, Juan, and Wendy. I want to thank my family. My mother, who traveled back and forward from my home county, every year for eight years, to help me taking care of my children which was essential to me during my graduate studies. Without her help I would not have had peace of mind during my long lab hours. I thank my cousin Melisa who entertained my energetic younger son allowing me to write my thesis. I especially want to thank my husband Luis. He was my rock during all the challenging circumstances that arose. He supported and encouraged me and would always be there to remind me that I could achieve my goals and that he would do everything he could to help me make it possible. His positivity and love were essential for me during the most difficult times. I also thank him for being the great father that he is and for making sure the kids had all they needed when I could not be there during my long days in the lab. I thank my son Tomas who most of the time was able to patiently wait for me to be able to spend time with him. Although I stretched his patience to the limit he always gave me a smile and a hug and reminded me that he was there for me and loved me very much.