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Study of Ascl1 Function in the Neurogenic Lineage of the Adult Mouse Hippocampus

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Study of Ascl1 Function in the Neurogenic Lineage of the Adult Mouse Hippocampus Study of Ascl1 function in the neurogenic lineage of the adult mouse hippocampus Jimena Andersen March 2015 Division of Molecular Neurobiology MRC National Institute for Medical Research The Ridgeway, Mill Hill, London NW7 1AA Department of Cell and Developmental Biology University College London Thesis submitted to University College London for the degree of Doctor of Philosophy 1 Declaration of authenticity The work presented in this thesis has been completed in the laboratory of François Guillemot in the Division of Molecular Neurobiology at the National Institute for Medical Research, currently The Francis Crick Institute, Mill Hill Laboratory. I, Jimena Andersen, confirm that this work is my own. Where information has been derived from other sources, this has been indicated in the thesis. Processing of tissues for immunohistochemical analysis (perfusion, vibratome sectioning and staining) was done in its majority by Angeliki Achimastou (former research assistant in the laboratory). Laser capture microdissection (LCM) analysis in the Results sections 3.3.2, 4.3.2 and 4.3.4 was performed by Ayako Ito (former post-doc in the laboratory). ChIP-seq analysis referred to in Chapter 6 and described in Appendix 1 was performed by Ben Martynoga (former post-doc in the laboratory). Finally, isolation of RGLs by FACS was done in collaboration with Noelia Urbán (current post-doc in the laboratory). 2 Abstract The adult mammalian brain is a highly plastic structure capable of cellular and molecular remodelling in response to its interactions with the outside world. The addition of new neurons to the hippocampus throughout life is one of the most striking manifestations of this plasticity. New neurons here are generated from a population of stem cells that, although existing primarily in a dormant or quiescent state, they can become activated upon the reception of neurogenic signals. How stem cells integrate these signals from the environment to ultimately control neuronal production is currently under investigation. During embryonic development, transcription factors of the basic helix-loop- helix family promote progenitor proliferation and differentiation to ensure the production of neurons in correct numbers and at the correct positions. We found Ascl1, a proneural factor in this family, to be expressed by stem cells of the adult hippocampus when in an active state. Here we used pharmacological and genetic approaches to show that Ascl1 expression is rapidly induced in response to neurogenic stimuli, and that deletion of this factor with a conditional inactivation approach results in an inability of stem cells to respond to signals and exit their quiescent state. Moreover, by examining the genes deregulated in Ascl1-deleted stem cells, we show that Ascl1 promotes the proliferation of hippocampal stem cells by directly regulating cell cycle regulatory genes, among which the cyclin D genes are of great importance. The data presented here supports a model whereby Ascl1 acts as a central factor in adult hippocampal stem cells to integrate both stimulatory and inhibitory signals and translate them into a transcriptional programme that controls stem cell activity. With this work we also highlight that understanding how Ascl1 is regulated will contribute, in the future, to the development of stem cell therapies for the treatment of neurological disorders. 3 Table of contents Abstract ....................................................................................................................... 3 List of Figures and Tables ......................................................................................... 9 Abbreviations ........................................................................................................... 13 Acknowledgments .................................................................................................... 17 Chapter 1 Introduction ........................................................................................... 19 1.1 An introduction to the field ........................................................................... 21 1.1.1 Brief historical perspective .................................................................... 21 1.1.2 Definitions: What is adult neurogenesis? ............................................... 22 1.1.3 Neurogenesis in the adult human brain .................................................. 24 1.1.4 An evolutionary view of adult neurogenesis .......................................... 25 1.2 Neurogenic niches in the adult brain ............................................................. 26 1.2.1 The SVZ neurogenic niche .................................................................... 27 1.2.2 The DG neurogenic niche ...................................................................... 29 1.3 The adult mouse hippocampus ...................................................................... 31 1.3.1 Neuronal circuits in the adult hippocampus ........................................... 32 1.3.2 Developmental origin of the hippocampal DG ...................................... 33 1.3.3 Neuronal development in the adult DG ................................................. 34 1.3.4 Regulation of adult neurogenesis in the hippocampus ........................... 39 1.3.4.1 Adult hippocampal neurogenesis and ageing .................................. 44 1.3.5 Functional significance of adult hippocampal neurogenesis ................. 45 1.3.6 Adult neurogenesis and neurological disorders ..................................... 47 1.4 Mechanisms involved in the control of adult hippocampal neurogenesis .... 49 1.4.1 Mechanisms involved in the maintenance of quiescence ...................... 50 1.4.1.1 Extrinsic factors .............................................................................. 50 4 1.4.1.2 Intrinsic factors ............................................................................... 51 1.4.2 Mechanisms involved in stem cell and progenitor proliferation ............ 52 1.4.2.1 Extrinsic factors .............................................................................. 52 1.4.2.2 Intrinsic factors ............................................................................... 54 1.5 The proneural factor Ascl1 in neurogenesis ................................................. 56 1.5.1 Proneural factors and their mechanism of action ................................... 56 1.5.2 Ascl1 during cortical development ........................................................ 58 1.5.3 Ascl1 in the adult brain .......................................................................... 60 1.6 Aims of the present work .............................................................................. 61 Chapter 2 Materials and Methods ......................................................................... 63 2.1 Animals ......................................................................................................... 64 2.2 Genotyping .................................................................................................... 64 2.3 In vivo studies: .............................................................................................. 65 2.3.1 Tamoxifen and BrdU administration ..................................................... 65 2.3.2 Voluntary running .................................................................................. 66 2.3.3 Social isolation ....................................................................................... 66 2.3.4 Kainic acid administration ..................................................................... 66 2.4 Expression pattern analysis ........................................................................... 67 2.4.1 RNA probes ............................................................................................ 67 2.4.2 Tissue preparation .................................................................................. 67 2.4.3 Immunohistochemistry ........................................................................... 68 2.4.4 Microscopic analysis and quantification ................................................ 70 2.4.5 In situ hybridisation ............................................................................... 70 2.5 Fluorescence-activated sell sorting ............................................................... 71 2.6 Laser capture microdissection ....................................................................... 71 2.7 Gene expression assays ................................................................................. 72 5 2.7.1 RNA isolation ........................................................................................ 72 2.7.2 cDNA production ................................................................................... 72 2.7.3 Quantitative-PCR ................................................................................... 72 2.8 Statistical and bioinformatics analyses ......................................................... 73 Chapter 3 Results: Ascl1 deletion in stem cells of the adult dentate gyrus ........ 74 3.1 Ascl1 is expressed in intermediate progenitors ............................................. 75 3.2 Ascl1 is expressed in a subset of stem cells .................................................. 77 3.3 Ascl1 deletion in stem cells of the adult hippocampus ................................. 77 3.3.1 Mouse lines used for deleting Ascl1 ...................................................... 77 3.3.2
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