PLEIOTROPIC AND ISOFORM-SPECIFIC FUNCTIONS OF PITX2 IN BRAIN DEVELOPMENT by Mindy Rachelle Waite A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Cellular and Molecular Biology) in The University of Michigan 2012 Doctoral Committee: Associate Professor Donna M. Martin, Chair Professor Gregory R. Dressler Professor Sue O’Shea Associate Professor Philip J. Gage Associate Professor Robert C. Thompson Yesterday is but a dream, tomorrow but a vision. But today well lived makes every yesterday a dream of happiness, and every tomorrow a vision of hope. Look well, therefore, to This Day. -Sanskrit Proverb © Mindy Rachelle Waite 2012 ACKNOWLEDGMENTS I would like to thank Dr. Donna Martin for her unwavering mentorship and support. Donna, I can never repay you enough for taking me in as one of your own and building my skillset from the ground up. You really put your heart and soul into teaching me how to do research, write, speak, and even work in a team setting. Thank you so much for the skills and self-confidence which will be necessary for my future successes. I will never forget our first “data dance” and promise to think of you every time I see red ink. I would also like to thank the rest of my lab. Liz, Jennifer, Joe, Wanda, Kaia, and Jillian – what would I have ever done without you guys? Graduate school can be a frustrating, lonely experience for some, but not for me. Every day I worked with my friends. Thank you for your unconditional support and delightful shenanigans. I will miss you all dearly and wish upon you success and happiness. I sincerely hope that we work together again- probably because “I love you”. I am eternally grateful to the members of my esteemed thesis committee, Donna Martin, Gregory Dressler, Phil Gage, Sue O’Shea, and Bob Thompson who were thoughtful, candid, insightful, and even kind. Your guidance was absolutely critical for the progress of my studies as well as my professional development. Thank you for having my best interests at heart from day one through all these years. Thanks to the Program in Biomedical Sciences (PiBS) and the Cellular and Molecular Biology Program (CMB) at the University of Michigan for giving me extraordinary learning opportunities at the bench and outside of the laboratory. I hope to make your proud in my future interdisciplinary endeavors. Special thanks to Jessica Schwartz, Cathy Mitchell, Andrzej Dlugosz, Kate Barald, and Deborah Gumucio with whom I have worked closely or leaned upon during my graduate career. ii To my fellow PiBS colleagues- thank you for your scientific (and non-scientific) discussions, lending of reagents, feedback, support, and especially for your friendship. I am deeply thankful to Jeff Calhoun, Ilea Swineheart, Lauren Van Wassenhove, Jill Haenfler, Jamie Lane, and Krista Geister. I know that our careers may diverge from here, but I look forward to seeing where we all end up. Go blue! I would like to thank my family members for their support (especially the care packages). In the last five years, I have grown a lot and followed new and unexplored passions. Thank you for at least trying to accept the person I have become and the unique paths I have chosen. Are you ready for another adventure? Finally, I acknowledge the miracles performed by my husband, Joseph Munski. I (and my work) have benefited greatly from his insightful conversations, motivational pep talks, emotional support, domestic capabilities, and his can-do attitude. Thank you for listening to my practice talks, editing my papers, and even coming in to lab with me for motivation. Joseph, this journey would have been a much greater struggle without you and I’m thrilled that you came along for the ride! P.S. Pterodactyl. iii Table of Contents Acknowledgements…………………………………………………………………….. ii List of Figures………………………………………………………………………….. v Abstract………………………………………………………………….…….……….. vii Chapter I. Introduction……………………………………………………………..…….. 1 II. Distinct populations of GABAergic neurons in mouse rhombomere 1 express but do not require the homeodomain transcription factor PITX2 Abstract…………………………………………………………………..… 35 Introduction………………………………………………………………… 36 Materials and Methods……………………………………………...……… 37 Results……………………………………………………………………… 39 Discussion……………………………………………………………..…… 45 Acknowledgements………………………………………………………… 50 References……………………………………………….…………………. 60 III. GABAergic and glutamatergic identities of developing midbrain Pitx2 neurons Abstract…………………………………………………….…….………… 65 Introduction………………………………………………………………… 66 Materials and Methods…………………………………………….……..… 67 Results……………………………………………………………………… 69 Discussion………………………………………………………………..… 76 Acknowledgements………………………………………………………… 80 References………………………………………………………….………. 95 IV. Pleiotropic and isoform-specific functions for Pitx2 in superior colliculus and hypothalamic neuronal development Abstract………………………………………………………………..…… 100 Introduction………………………………………………………………… 101 Materials and Methods……………………………………………..………. 103 Results……………………………………………………………………… 106 Discussion……………………………………………………….…….…… 112 Acknowledgements………………………………………………………… 118 References……………………………………………………..…………… 131 V. Conclusion Identities of CNS Pitx2-positive neurons………………………………….. 138 Axial-level and isoform-specific requirements for Pitx2 during brain development…………………………………………………………….….. 142 Summary………………………………………………………...…………. 144 References………………………………………………………………….. 146 Appendix……………………………………………………………………………….. 149 iv List of Figures Figure 1.1 PITX transcription factor family……………………………………….………. 17 1.2 Map of Pitx2 expression during mouse embryonic development…….……..…. 18 1.3 PITX2 gene and map of alternative splicing………………………………….... 19 1.4 Pitx2 employment in developmental…………………………………………... 20 1.5 Sagittal E14.5 mouse CNS map. ………………………………….…………… 21 2.1 Pitx2-expressing cells localize to rhombomere 1……………………………… 51 2.2 PITX2-positive GABAergic neurons in r1 are distinct from serotonergic, glutamatergic, and cholinergic neurons………………………………………... 52 2.3 GABAergic r1 neurons express PITX2, EN1, and LHX1/5…………………… 53 2.4 PITX2-positive GABAergic neurons occupy distinct regions of the ventral hindbrain……………………………………………………………………….. 54 2.5 Early expression of PITX2 is similar to E12.5 patterning and includes Dbx1-lineage cells……………………………………………………….…….. 55 2.6 PITX2 is dispensable for early neuronal migration and ventral r1 patterning………………………………………………………………………. 56 2.7 Two PITX2-positive populations span ventral r1……………………………… 57 2.8 Ventral r1 GABAergic identity is PITX2-independent……………….……….. 58 2.9 Schematic of transverse sections through the developing mouse brain at the level of rhombomere 1…………………………………………………... 59 3.1 PITX2 identifies GABAergic interneurons in an intermediate layer of the dorsal midbrain…………………………………………………………….. 82 3.2 Collicular glutamatergic neurons are BRN3A-positive and PITX2-negative…. 84 3.3 PITX2-positive cells represent a unique population of GABAergic dorsal midbrain precursors……………………………………………………………. 85 3.4 PITX2 is required for GABAergic differentiation…………………………….. 86 3.5 PITX2 identifies restricted populations of ventromedial midbrain precursors… 87 3.6 Ventral midbrain domains are delineated by transcription factor patterning….. 88 3.7 Early ventral midbrain patterning is PITX2-independent……………………… 89 3.8 Pitx2-lineage neurons are glutamatergic and sparse in the red nucleus….…… 90 3.9 Pitx2 expression is transient in the ventral midbrain…………………………... 91 3.10 Summary of Pitx2 expression in the developing dorsal and ventral midbrain……………………………………………………………….……….. 92 3.11 Supplementary Figure S1. Early rostral-caudal patterning of the midbrain is PITX2-independent…………………………………………………….……. 94 4.1. Pitx2 isoforms and alleles……………………………………………………… 119 4.2. Pitx2 is expressed in early post-mitotic midbrain neurons…………………….. 121 v 4.3. Pitx2ab and Pitx2c are expressed in midbrain neurons………………………... 122 4.4. Pitx2 isoforms exhibit differential contributions to midbrain neuron migration……………………………………………………………….. 124 4.5. Pitx2ab regulates the timing of midbrain neuronal migration…………...…….. 125 4.6. Evidence for cell-autonomous effects of Pitx2 deficiency on collicular neuronal migration…………………………………………………………….. 126 4.7. Collicular GABAergic differentiation requires a single allele dose of either Pitx2ab or Pitx2c…………………………………….………………………….127 4.8. PITX2AB is necessary for formation of the mammillothalamic tract (MTT)…. 129 A.1. Pitx2 is dispensable for early patterning of migrational markers…………..….. 154 A.2. Pitx2 is required for proliferation of midbrain progenitors……………………. 155 A.3. Pitx2 positively regulates midbrain progenitor proliferation through non tissue-autonomous mechanisms…………………………………………… 156 vi ABSTRACT Pleiotropic and isoform-specific functions of PITX2 in brain development by Mindy Rachelle Waite Chair: Donna M. Martin Neuronal diversification in the developing brain is a coordinated process requiring complex genetic regulation. Transcriptional control of gene expression is known to regulate proliferation, migration, differentiation, and survival of developing neurons, although the precise mechanisms underlying these processes are not fully understood. One transcription factor, pituitary homeobox 2 (Pitx2), is expressed in the developing and adult mammalian brain, eye, and pituitary, and in asymmetric organs such as the heart, lungs, and gut. In humans, mutations in PITX2 result in the autosomal dominant disorder Rieger syndrome which is characterized by defects in