The cellular and molecular consequences of maternal immune activation on the cytoarchitecture of the developing spinal cord Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy Rebecca C Anderson Supervisor: Professor Kieran McDermott Co-Supervisor: Dr Gerard O’Keeffe February 2020 Table of Contents: Declaration: ........................................................................................................................ i Acknowledgements: .......................................................................................................... ii Abstract: ............................................................................................................................ v Abbreviations: ................................................................................................................ viii Table of Figures: ............................................................................................................. xv Table of Tables:............................................................................................................ xviii Chapter 1. Introduction .................................................................................................. 1 1.1 Nervous system development ...................................................................................... 2 1.1.1 Neurulation ...................................................................................................................... 2 1.1.2 Cell signalling and nervous system development ............................................................ 4 1.1.2.1 Stem cell renewal and pluripotency .......................................................................... 4 1.1.2.2 Migration ................................................................................................................... 5 1.1.2.4 Cytokines, signalling, and nervous system development .......................................... 7 1.1.3 Spinal cord development & anatomy ............................................................................... 8 1.2. Cellular development ............................................................................................... 11 1.2.1 Neurogenesis ................................................................................................................. 11 1.2.2 Oligodendrogliogenesis .......................................................................................... 13 1.2.3 Astrogliogenesis ...................................................................................................... 17 1.2.4 Microgliogenesis ..................................................................................................... 19 1.3 Maternal immune activation ..................................................................................... 22 1.3.1 Epidemiological studies ................................................................................................. 22 1.3.2 Animal models ............................................................................................................... 24 1.3.2.1 Polyinosinic:polycytidylic acid ............................................................................... 24 1.3.2.2 Lipopolysaccharide ................................................................................................. 24 1.3.2.3 Other models of maternal infection......................................................................... 25 1.3.3 The rodent spinal cord as a model of MIA .................................................................... 25 1.3.4 MIA and Neurons .......................................................................................................... 26 1.3.5 MIA and oligodendrocytes............................................................................................. 31 1.3.5.1 Cytokines, inflammation and oligodendrocyte development .................................. 31 1.3.5.2 The consequences of MIA on the developing oligodendrocyte .............................. 32 1.3.6 MIA and Astrocytes ....................................................................................................... 37 1.3.6.1 The role of astrocytes in mediating the consequences of MIA ............................... 37 1.3.6.2 The consequences of MIA on the developing astrocyte ......................................... 37 1.3.7 MIA and Microglia ........................................................................................................ 40 1.3.7.1 Cytokines, inflammation and microglial development ........................................... 40 1.3.7.2 The consequences of MIA on the developing microglia......................................... 41 1.4 MicroRNAs ............................................................................................................... 44 1.4.1 Structure and function of microRNAs ........................................................................... 45 1.4.2 Nomenclature ................................................................................................................. 46 1.4.3 MicroRNAs and cellular development .......................................................................... 47 1.4.3.1 MicroRNAs in neurogenesis ................................................................................... 47 1.4.3.2 MicroRNAs in astrogliogenesis .............................................................................. 49 1.4.3.3 MicroRNAs in microgliogenesis............................................................................. 50 1.4.3.4 MicroRNAs in Oligodendrogliogenesis .................................................................. 50 1.5 Aims and objectives: ................................................................................................. 51 Chapter 2. Methodology ............................................................................................... 52 2.1 Time mating and maternal immune activation.......................................................... 53 2.2 Tissue collection and processing ............................................................................... 54 2.2.1 Tissue collection for embryonic study ........................................................................... 54 2.2.2 Tissue collection for postnatal study .............................................................................. 54 2.2.3 Gelation coating of slides ............................................................................................... 55 2.2.4 Cryosectioning ............................................................................................................... 55 2.3 Immunohistochemical staining ................................................................................. 57 2.3.1 Immunofluorescent staining ........................................................................................... 57 2.3.2 Quantification of immunofluorescent staining............................................................... 60 2.3.2.1 Olig2 analysis .......................................................................................................... 61 2.3.2.2 MBP ........................................................................................................................ 63 2.3.2.3 Iba-1 ........................................................................................................................ 64 2.3.2.4 GFAP ...................................................................................................................... 64 2.3.2.5 Reelin ...................................................................................................................... 65 2.4 RNA extraction and yield determination .................................................................. 66 2.4.1 Total RNA extraction ..................................................................................................... 66 2.4.2 Quality and yield determination ..................................................................................... 66 2.5 RNA expression studies ............................................................................................ 67 2.5.1 Microarray expression profiling..................................................................................... 67 2.5.2 RNASeq ......................................................................................................................... 68 2.5.3 RT qPCR ASSAY .......................................................................................................... 68 2.6 Bioinformatic analysis .............................................................................................. 71 2.6.1 Literature review ............................................................................................................ 71 2.6.2 miRECORDS ................................................................................................................. 71 2.6.3 Webgestalt...................................................................................................................... 72 2.6.4 String analysis ................................................................................................................ 72 Chapter 3. The effect of MIA on the oligodendrocytes of the spinal cord ............... 74 3.1 Chapter Abstract........................................................................................................ 75 3.2 Introduction ..............................................................................................................