THE ROLE OF MICRORNA-9 IN VERTEBRATE NEURAL DEVELOPMENT A thesis submitted to the University of Manchester for the Wellcome Trust 4 Year PhD in the Faculty of Life Sciences 2011 Boyan Bonev Table of Contents TITLE PAGE .......................................................................................................................................... 1 TABLE OF CONTENTS ......................................................................................................................... 2 TABLE OF FIGURES ............................................................................................................................. 3 ABSTRACT ............................................................................................................................................. 5 ACKNOWLEDGEMENTS ..................................................................................................................... 7 ABBREVIATIONS .................................................................................................................................. 8 RATIONALE FOR SUBMITTING IN ALTERNATIVE FORMAT .................................................. 9 AUTHOR CONTRIBUTIONS ............................................................................................................ 10 CHAPTER 1. INTRODUCTION ........................................................................................................ 11 1. DEVELOPMENT OF THE VERTEBRATE CENTRAL NERVOUS SYSTEM .................................................. 11 1.1. Extrinsic signals regulating neuronal differentiation ....................................................... 13 1.1.1. Wnt signaling .............................................................................................................................................................. 13 1.1.2. Fibroblast Growth Factor (FGF) signaling ..................................................................................................... 14 1.1.3. Notch signaling .......................................................................................................................................................... 16 1.2. Intrinsic mechanisms of cell fate regulation during neural development ................ 17 1.2.1. Chromatin state determines the probability of transcription ............................................................... 18 1.2.2. DNA methylation ....................................................................................................................................................... 19 2. MORPHOLOGICAL CHANGES DURING TERMINAL NEURONAL DIFFERENTIATION ............................ 20 2.1.1. Axon specification .................................................................................................................................................... 20 2.1.2. Axon elongation ......................................................................................................................................................... 21 2.1.1. Axon branching .......................................................................................................................................................... 22 2.1.2. Local control on axonal mRNA translation .................................................................................................... 23 3. MICRORNAS IN NEURAL DEVELOPMENT ............................................................................................... 24 3.1. Biogenesis, mechanism and function ........................................................................................ 24 3.2. microRNA function in the development of the nervous system ..................................... 27 3.3. miRNA-9 – an important regulator of vertebrate neural development .................... 29 AIMS AND OBJECTIVES ................................................................................................................... 31 2 CHAPTER 2. MICRORNA-9 REVEALS REGIONAL DIVERSITY OF NEURAL PROGENITORS ALONG THE ANTERIOR-POSTERIOR AXIS .................................................. 33 CHAPTER 3. MICRORNA-9 MODULATES HES1 ULTRADIAN OSCILLATIONS BY FORMING A DOUBLE NEGATIVE FEEDBACK LOOP ................................................................ 34 CHAPTER 4. MICRORNA-9 REGULATES AXON EXTENSION AND BRANCHING BY TARGETING MAP1B IN MOUSE CORTICAL NEURONS ........................................................... 61 CHAPTER 5. DISCUSSION ............................................................................................................... 93 REFERENCES .................................................................................................................................... 104 Total word count (excluding publications): 26,796 3 Table of Figures FIGURE 1. CELL TYPES COMPRISING THE VERTEBRATE CENTRAL NERVOUS SYSTEM ....................... 12 FIGURE 2. WNT SIGNALING IN NEURAL DEVELOPMENT. ...................................................................... 14 FIGURE 3. FGF8 SIGNALING IN NEURAL DEVELOPMENT ...................................................................... 15 FIGURE 4. NOTCH SIGNALING GENERATES HES1 MOLECULAR OSCILLATIONS ................................. 16 FIGURE 5. SPECIFICATION AND ELONGATION OF THE AXON DURING NEURONAL MATURATION ... 21 FIGURE 6. AXON BRANCHING IS AN ESSENTIAL STEP DURING NEURONAL MATURATION. ............... 23 FIGURE 7. BIOGENESIS OF MICRORNAS. .............................................................................................. 25 FIGURE 8. MICRORNAS MECHANISMS OF TARGET RECOGNITION AND SILENCING ........................ 26 4 ABSTRACT The University of Manchester Boyan Bonev Wellcome Trust 4 Year PhD Thesis Title: The Role of microRNA-9 in vertebrate neural development Date: 27-09-2011 During neural development proliferating cells in the ventricular Zone undergo repeated self-renewal to maintain the progenitor pool or, alternatively exit the cell cycle and differentiate into neurons. This process is regulated by the coordination of cell intrinsic signals and regional and temporal specific external cues which determine the type and the amount of neurons generated. After a neural progenitor has committed to differentiation the process of balancing internal and external signals continues during maturation to guide the initiation, elongation and branching of the axon. One major remaining question is how biological regulation can be integrated with the developmental context to produce a specific outcome. Here, I show that microRNAs and particularly, microRNA-9 (miR-9) plays a very important role in vertebrate neural development; a role, which is highly dependent on the context. In X. tropicalis, miR-9 reveals regional specific progenitor heterogeneity – it is required for cell cycle exit and differentiation throughout the neural tube, but forebrain progenitors additionally, and uniquely, require miR-9 for their survival. I have shown that the major miR-9 target in this context is the hairy and enhancer of split gene hairy1. When miR-9 is absent, hairy1 domain expands and selectively activates different signaling pathways in the forebrain and the hindbrain, culminating in the regional specific differences we observed. In the mouse, the homologue of hairy1 – Hes1 expression is oscillatory which is essential for progenitor maintenance. I have shown that Hes1 is also a subject of miR-9 regulation, which, in this context, is necessary for maintaining the oscillations. Furthermore, miR-9 is also regulated by Hes1, which leads to opposite-phase oscillations of miR-9 primary transcripts, but step-wise accumulation of the mature miR-9 form due to its stability. I propose that miR-9 levels act as output to measure the number of Hes1 cycles and at certain critical threshold levels this leads to dampening of the oscillations and allows progenitors to differentiate. MiR-9 is also expressed in differentiated neurons in the forebrain, where its function is completely unknown. We have shown that in this context it promotes axon branching and inhibits axon growth through the microtubule-associated protein 1B (MAP1B). We have also provided evidence that brain-derived neurotrophic factor (BDNF) can modulate this process by regulating miR-9 levels in the axon. Overall, these findings contribute to our understanding of neural development and provide novel explanation of how to cell fate decisions are integrated with context-specific signals to generate a specific outcome. 5 DECLARATION No portion of the work referred to in the thesis has been submitted in support of an application for another degree or qualification of this or any other university or other institute of learning. 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