Mechanisms of Brain Ventricle Development
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Mechanisms of Brain Ventricle Development by Laura Anne Lowery B.S. Biology University of California San Diego, 2000 M.S. Biology University of California San Diego, 2001 Submitted to the Department of Biology in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Biology at the Massachusetts Institute of Technology June 2008 © 2008 Laura Anne Lowery. All rights reserved. The author hereby grants to MIT permission to reproduce and to distribute publicly paper and electronic copies of this thesis document in whole or in part. Signature of Author…………….…………………………………………………………… Department of Biology May 2008 Certified by…….…………………………………………………………………………… Hazel L. Sive Professor of Biology Associate Dean School of Science Thesis Supervisor Accepted by.………………………………………………………...……………………… Stephen P. Bell Professor of Biology Chairman, Graduate Student Committee 1 2 Mechanisms of Brain Ventricle Formation By Laura Anne Lowery Submitted to the Department of Biology on April 2008 in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Biology ABSTRACT The brain ventricles are a conserved system of fluid-filled cavities within the brain that form during the earliest stages of brain development. Abnormal brain ventricle development has been correlated with neurodevelopmental disorders including hydrocephalus and schizophrenia. The mechanisms which regulate formation of the brain ventricles and the embryonic cerebrospinal fluid are poorly understood. Using the zebrafish, I initiated a study of brain ventricle development to define the genes required for this process. The zebrafish neural tube expands into the forebrain, midbrain, and hindbrain ventricles rapidly, over a four-hour window during mid-somitogenesis. In order to determine the genetic mechanisms that affect brain ventricle development, I studied 17 mutants previously-identified as having embryonic brain morphology defects and identified 3 additional brain ventricle mutants in a retroviral-insertion shelf-screen. Characterization of these mutants highlighted several processes involved in brain ventricle development, including cell proliferation, neuroepithelial shape changes (requiring epithelial integrity, cytoskeletal dynamics, and extracellular matrix function), embryonic cerebrospinal fluid secretion, and neuronal development. In particular, I investigated the role of the Na+K+ATPase alpha subunit, Atp1a1, in brain ventricle formation, elucidating novel roles for its function during brain development. This study was facilitated by the snakehead mutant, which has a mutation in the atp1a1 gene and undergoes normal brain ventricle morphogenesis but lacks ventricle inflation. Analysis of the temporal and spatial requirements of atp1a1 revealed an early requirement during formation, but not maintenance, of the neuroepithelium. I also demonstrated a later neuroepithelial requirement for Atp1a1-driven ion pumping that leads to brain ventricle inflation, likely by forming an osmotic gradient that drives fluid flow into the ventricle space. Moreover, I have discovered that the forebrain ventricle is particularly sensitive to Na+K+ATPase function, and reducing or increasing Atp1a1 levels leads to a corresponding decrease or increase in ventricle size. Intriguingly, the Na+K+ATPase beta subunit atp1b3a, expressed in the forebrain and midbrain, is specifically required for their inflation, and thus may highlight a distinct regulatory mechanism for the forebrain and midbrain ventricles. In conclusion, my work has begun to define the complex mechanisms governing brain ventricle development, and I suggest that these mechanisms are conserved throughout the vertebrates. Thesis Supervisor: Hazel L. Sive Title: Professor of Biology 3 4 Dedicated to my grandparents, Lester and Leah Sopkin 5 6 Table of Contents Page Abstract 3 Acknowledgements 9 Curriculum Vitae 11 Chapter 1 Introduction – A Discussion of the Formation and Function of the Embryonic Brain Ventricles 13 Introduction 15 The Brain Ventricular System 16 Brain Ventricle Abnormalities 18 Formation of the Embryonic Brain Ventricles 20 Mechanisms of Brain Ventricle Development 21 Preview of Thesis 34 Figures 35 References 51 Chapter 2 The zebrafish as a model for analyzing neural tube defects 63 Introduction 65 Zebrafish as a Model System 65 Ontogeny of the Zebrafish Nervous System 67 Zebrafish as a Model for Brain Ventricle Formation 75 Lessons from Zebrafish Mutants 78 Conclusion 82 Table and Figures 83 References 99 Chapter 3 Characterization and Classification of 20 Zebrafish Brain Morphology Mutants 111 Abstract 113 Background 114 Results and Discussion 115 Conclusions 123 Experimental Procedures 124 Table and Figures 127 References 141 7 Chapter 4 Initial Formation of Zebrafish Brain Ventricles Occurs Independently of Circulation and Requires the nagie oko and snakehead/atp1a1 Gene Products 147 Abstract 149 Introduction 150 Results 151 Discussion 158 Experimental Procedures 161 Figures 167 References 187 Chapter 5 The Spatial and Temporal Requirements for Na+K+ATPase during Brain Ventricle Development 193 Abstract 195 Introduction 196 Results 197 Discussion 204 Experimental Procedures 207 Tables and Figures 213 References 233 Chapter 6 Conclusions and Future Directions 239 Future studies of eCSF formation and function 241 Na+K+ATPase function during brain development 246 Cell proliferation and brain ventricle opening 248 Table and Figures 249 References 261 Appendix 1 whitesnake/sfpq is Required for Cell Survival and Neuronal Development in the Zebrafish 265 Abstract 266 Introduction 267 Results and Discussion 267 Experimental Procedures 274 Figures 279 References 301 Appendix 2 Formation of the midbrain-hindbrain boundary constriction requires laminin-dependent basal constriction 305 Abstract 306 Introduction 307 Results and Discussion 308 Experimental Procedures 312 Figures 315 References 327 8 Acknowledgments I would like to thank all members of the Sive lab for their encouragement, support, and advice over the past six years. I am particularly thankful to the generosity of Elizabeth Wiellette, who taught me almost everything related to the zebrafish, Amanda Dickinson, who taught me microscopy, imaging, and Photoshop, and Jennifer Gutzman, my baymate and partner in zebrafish brain morphogenesis for the last three years. Jen Gutzman, in particular, has generously answered countless questions and has always been willing to give me needed feedback. Additionally, Jen Gutzman and Ellie Graeden provided significant support editing several thesis chapters. Furthermore, I could not have done my research without the fish husbandry provided by the Sive lab fish technician, Olivier Paugois. It was also a pleasure to mentor two MIT undergraduates, Jamie Rubin and Jenny Ruan, and I enjoyed teaching them as well as benefitted from their learning. The Sive Lab is fortunate to have the incredible Heather Ferguson as our administrative assistant, and I am quite appreciative of her work. Finally, I am indebted to my advisor, Hazel Sive. Without a doubt, I could not have chosen a better graduate advisor. She has been extraordinarily supportive, challenging, and understanding, changing depending on my own needs, and my success is in part a reflection of her outstanding abilities as my advisor. I will always be grateful for her infallible support. I would also like to thank the current and past members of my thesis committee for their time, energy, and support: Martha Constantine-Paton, Richard Hynes, Paul Garrity, David Housman, and Michael Levin. My research project could not have been possible without the great generosity of the zebrafish community. Over a dozen different labs provided me with zebrafish mutants to study and other useful reagents. Additionally, I want to thank Duaa Mohammad for friendship and support throughout graduate school. She also read parts of this thesis and provided critiques. Most importantly, I need to thank my family: my parents, who taught me that I have the ability to do anything and who have always believed in me; my in-laws Catherine and Burrell, who provided me with a special home and refuge during my time here at MIT; my son, Elliot, who has reminded me in an extreme way that there are more important things in life than work; and I am most thankful to my husband, Drew, who has brought me peace in times of chaos and has provided incredible support and partnership throughout graduate school and throughout our life together. 9 10 Curriculum Vitae Laura Anne Lowery former name: Laura Anne Hardaker Education • Ph.D. Biology, Massachusetts Institute of Technology – 06/08 • M.S. Biology, University of California, San Diego – 03/01 • B.S. Biology, University of California, San Diego – 03/00, Cum Laude Research Experience • 05/02 – 06/08, Doctoral thesis in lab of Dr. Hazel Sive, MIT/Whitehead Institute “Mechanisms of brain ventricle development” • 04/99-06/01, Master’s thesis in lab of Dr. William R. Schafer, UC San Diego “Neuroendocrine regulation of egg-laying behavior in C. elegans” Teaching Experience • Fall 04 – Spring 06: Whitehead High School Partnership Program • Fall 04 – Spring 07: Undergraduate Research Mentor, MIT • Spring 05: Graduate Teaching Assistant, 7.02: Undergrad Biology Lab, MIT • Fall 02: Grad Teaching Assistant, 7.22: Undergrad Developmental Biology, MIT • 11/00: Guest Lecturer for Undergraduate Biology class, University of San Diego • Spring 00: Grad Teaching Assistant, BIBC 102: Undergrad Metabolic Biochemistry, UCSD • Winter 00: Undergrad