I have sought to discover genetic causes of neuroanatomical defects by conducting N-ethyl-N-nitrosourea mutagenesis and transgenic knock out screens in mice. The rationale behind this is that mutations causal to structural defects will be informative about developmental neurobiology and the biological basis of behaviour. Direct screening for behavioural abnormalities in mice has historically been arduous and yielded few findings due to small effect sizes and limited statistical power. My approach sought to bypass these problems by screening for highly penetrant morphological phenotypes. This thesis details my screens and the histological, genetic and behavioural characterisation of lines of interest. These include models of hydrocephalous, pyramidal cell layer ectopia, abnormal neurogenesis, corpus callosum agenesis, hippocampal enlargement, elevated cell death and hypomyelination. Whilst N-ethyl-N-nitrosourea mutagenesis screening has been conducted since the twilight of the 20^th century, systematic transgenic knock out screening is currently in its infancy. By discovering gene-phenotype associations through both approaches, I have been able to compare the relative yields, strengths and weaknesses of the two screening methods. Additionally, I have discussed the significant of the gene-phenotype associations produced from both screens.

Neurogenesis continues postnatally in two major neural stem cell (NSC) niches: The subventricular zone (SVZ) and dentate gurus of the hippocampus. SVZ NSCs self-renew and produce transit amplifying progenitor cells that, in turn, divide and give rise to neuroblasts. These neuroblasts migrate to the olfactory bulbs, via the rostral migratory stream (RMS), where they terminally differentiate into mature neurons. The postnatal SVZ (pSVZ) is more gliogenic than its adult counterpart (aSVZ), contributing to robust postnatal astrocytogenesis and oligodendrogenesis in the surrounding brain parenchyma. Studies examining Galectin-3 (Gal-3) in the aSVZ showed it has functions in regulating neuroblast migration, microglial activation, oligodendrocytic differentiation, and angiogenesis. However, the role of Gal-3 in pSVZ lineage progression is unknown. This thesis aims to unravel the roles of Gal-3 in regulating pSVZ lineage progression, fate choices, and NSC activation. In doing so, the thesis tackles the molecular pathways possibly involved in mediating the effects of Gal-3. I found through co-immunoprecipitation that Gal-3 was bound to β-catenin and both proteins were co-expressed in the aSVZ. In addition, expression of Gal-3 and Wnt/β-catenin signalling were downregulated as SVZ cells progressed through the lineage and became migratory. I hypothesised that Gal-3 may regulate lineage progression through regulation of Wnt/β-catenin signalling. To explore this hypothesis, Gal-3 overexpression, knockdown or control plasmids were co-electroporated with a Wnt/β-catenin reporter into the SVZ of postnatal day two mice. I found lineage progression was not altered by Gal-3 overexpression. Surprisingly, contrary to evidence described in the cancer literature, Gal-3 overexpression reduced Wnt/β-catenin signalling. This was accompanied by an acute reduction in proliferation. Also, more cells expressed p27/Kip1 in the SVZ, and more cells migrated into the RMS, suggesting increased cell cycle exit. However, NSC proliferation and clonal neurosphere forming capacity were not altered by Gal-3 overexpression, indicating that NSC activation was not influenced by Gal-3. While olfactory neuronogenesis was not altered by Gal-3 overexpression, striatal astrocytogenesis was increased while oligodendrogenesis was dampened. Further experiments revealed phosphorylation of Smad proteins 1/5/8 was increased in vivo and in vitro after Gal-3 overexpression. These findings indicate that Gal-3 positively regulated BMP signalling in the SVZ, possibly contributing to Gal-3's pro-gliogenic effects. Taken together, this thesis supports a model whereby a subpopulation of Gal-3-responsive pSVZ cells reacted to Gal-3 overexpression by acutely exiting the cell cycle, and possibly through the same mechanisms, switched from oligodendrocytic to astrocytic fate. These cellular responses might have been brought about, at least partially, by acute suppression of Wnt/β-catenin and activation of BMP signalling. These novel findings emphasise the regulatory actions of Gal-3 on pSVZ lineage progression through Wnt/β- catenin and BMP signalling.

<p dir="ltr">Primary cilia are hair-like structures found on nearly all mammalian cell types, including cells in the developing and adult brain. Cilia establish a unique signaling compartment for cells. For example, a diverse set of receptors and signaling proteins localize within cilia to regulate many physiological and developmental pathways including the Hh pathway. Defects in cilia structure, protein localization, or cilia function lead to genetic disorders called ciliopathies, which present with various clinical features including several neurodevelopmental phenotypes and hyperphagia associated obesity. Despite their dysfunction being implicated in several disease states, understanding their roles in CNS development and signaling has proven challenging. I hypothesize that dynamic changes to ciliary protein composition contributes to this challenge and may reflect unrecognized diversity of CNS cilia. The proteins ARL13B and ADCY3 are established ciliary proteins in the brain and assessing their localization is often used in the field to visualize cilia. ARL13B is a regulatory GTPase important for regulating cilia structure, protein trafficking, and Hh signaling, while ADCY3 is a ciliary adenylyl cyclase thought to be involved in ciliary GPCR singaling. Here, I examine the ciliary localization of ARL13B and ADCY3 in the perinatal and adult mouse brain by defining changes in the proportion of cilia enriched for ARL13B and ADCY3 depending on brain region and age. Furthermore, I identify distinct lengths of cilia within specific brain regions of male and female mice. As mice age, ARL13B cilia become relatively rare in many brain regions, including the hypothalamic feeding centers, while ADCY3 becomes a prominent cilia marker. It is important to understand the endogenous localization patterns of these proteins throughout development and under different physiological conditions as these common cilia markers may be more dynamic than initially expected. Understanding regional and development associated cilia signatures and physiological condition cilia dynamic changes in the CNS may reveal molecular mechanisms associated with ciliopathy clinical features such as obesity.</p>

Catechol-O-methyltransferase (COMT) metabolises catechol-containing compounds, including dopamine. The aim of this thesis was to investigate whether COMT is involved in hippocampal function. This thesis also explored the role of functional polymorphisms within the COMT gene in the pathogenesis of schizophrenia and schizophrenia-related phenotypes. First, as part of a study investigating the role of COMT in schizophrenia, human hippocampal COMT mRNA levels were shown to be neither altered in schizophrenia or bipolar disease, nor affected by COMT genotype. Hence, functional COMT polymorphisms do not appear to operate by altering gross COMT mRNA expression. Importantly, this study showed that COMT is expressed in the human hippocampus. Second, the role of COMT in hippocampal neurochemistry was explored by studying the effect of pharmacological COMT inhibition on catecholamines and metabolites in rat hippocampal homogenates, and extracellularly, using microdialysis. Both demonstrated that COMT modulates hippocampal dopamine metabolism. Thus, hippocampal COMT is of functional significance with respect to dopamine. Third, the effect of COMT inhibition on hippocampus-dependent behaviour was investigated. The results suggested a memory-enhancing effect of pharmacological COMT inhibition on hippocampus-dependent associative and non-associative forms of short-term memory in rats. In contrast, acute COMT inhibition appeared to have no effect on behavioural correlates of ventral hippocampal function i.e. anxiety-like behaviour. In summary, the expression of COMT mRNA in the human hippocampus, as well as the effect of COMT inhibition on rat hippocampal neurochemistry and hippocampus-dependent behaviour provide evidence for a functional role of COMT in the hippocampus. Moreover, changes in COMT activity alter hippocampal dopamine metabolism, which could be a potential mechanism for the role of COMT in hippocampus-dependent short-term memory.

The subplate layer of the cerebral cortex is comprised of a heterogeneous population of cells and contains some of the earliest-generated neurons. Subplate plays a fundamental role in cortical development. In the embryonic brain, subplate cells contribute to the guidance and areal targeting of corticofugal and thalamic axons. At later stages, these cells are involved in the maturation and plasticity of the cortical circuitry and the establishment of functional modules. In my thesis, I aimed to further characterize the embryonic murine subplate by establishing a gene expression profile of this population at embryonic day 15.5 (E15.5) using laser capture microdissection combined with microarrays. I found over 250 transcripts with presumed higher expression in the subplate at E15.5. Using quantitative RT-PCR, in situ hybridization and immunohistochemistry, I have confirmed specific expression in the E15.5 subplate for 13 selected genes which have not been previously associated with this compartment. In the reeler mutant, the expression pattern of a majority of these genes was shifted in accordance with the altered position of subplate cells. These genes belong to several functional groups and likely contribute to the maturation and electrophysiological properties of subplate cells and to axonal growth and guidance. The roles of two selected genes - cadherin 10 (Cdh10) and Unc5 homologue c (Unc5c) - were explored in more detail. Preliminary results suggest an involvement of Cdh10 in subplate layer organization while Unc5c could mediate the waiting period of subplate corticothalamic axons in the internal capsule. Finally, I compared the expression of a selection of subplate-specific genes (subplate markers) between mouse and rat and found some surprising species differences. Confirmed subplate markers were used to monitor subplate injury in a rat model of preterm hypoxiaischemia and it appeared that deep cortical layers including subplate showed an increased vulnerability over upper layers. Further characterization of subplate-specific genes will allow us to broaden our understanding of molecular mechanisms underlying subplate properties and functions in normal and pathological development.

Recent large-scale genome-wide studies for many human disorders have identified associations with numerous genetic variants. The biological interpretation of these variants presents a major challenge. In particular, the identification of biological pathways underlying the association could provide crucial insights into the disease aetiologies. In this thesis, I used functional genomics approaches to increase our understanding of neuropsychiatric and neurodevelopmental disorders. Firstly, in an integrative analysis of autism spectrum disorder (ASD), I looked into the role of genes targeted by Fragile-X Mental Retardation Protein ("FMRP targets"). I found evidence that FMRP targets contribute to ASD via two distinct aetiologies: (1) ultra-rare and highly penetrant single disruptions of embryonically upregulated FMRP targets ("single-hit aetiology") or (2) the combination of multiple less penetrant disruptions of synaptic FMRP targets ("multiple-hit aetiology"). In particular, I developed a pathway-association test sensitive to multiple-hit aetiologies. Secondly, I carried out an integrative analysis of bipolar disorder, following up a previously identified association with long-term potentiation. The association was not consistent across independent SNP and CNV datasets. Thirdly, I addressed the difficulty in identifying functional relationships between genes by integrating different datasets into a gene functional-linkage network tuned to the nervous system ("NsNet"). NsNet identified functional links between the genes disrupted by de novo loss-of-function mutations in ASD and, separately, in schizophrenia probands more sensitively than a general functional-linkage network. Fourthly, I considered the challenge of interpreting the phenotypic impact of gene disruptions, focusing on the identification of haploinsufficient genes. I constructed a gene haploinsufficiency score based on genome-wide datasets. Compared to existing approaches, the new score performed better in identifying less-studied haploinsufficient genes. This work both extends the methodology to detect the contribution of genetic variation to neuropsychiatric disorders and also yields insights into the variant genes and the pathways that underlie them. Firstly, in an integrative analysis of autism spectrum disorder (ASD), I looked into the role of genes targeted by Fragile-X Mental Retardation Protein ("FMRP targets"). I found evidence that FMRP targets contribute to ASD via two distinct aetiologies: (1) ultra-rare and highly penetrant single disruptions of embryonically upregulated FMRP targets ("single-hit aetiology") or (2) the combination of multiple less penetrant disruptions of synaptic FMRP targets ("multiple-hit aetiology"). In particular, I developed a pathway-association test sensitive to multiple-hit aetiologies. Secondly, I carried out an integrative analysis of bipolar disorder, following up a previously identified association with long-term potentiation. The association was not consistent across independent SNP and CNV datasets. Thirdly, I addressed the difficulty in identifying functional relationships between genes by integrating different datasets into a gene functional-linkage network tuned to the nervous system ("NsNet"). NsNet identified functional links between the genes disrupted by de novo loss-of-function mutations in ASD and, separately, in schizophrenia probands more sensitively than a general functional-linkage network. Fourthly, I considered the challenge of interpreting the phenotypic impact of gene disruptions, focusing on the identification of haploinsufficient genes. I constructed a gene haploinsufficiency score based on genome-wide datasets. Compared to existing approaches, the new score performed better in identifying less-studied haploinsufficient genes. This work both extends the methodology to detect the contribution of genetic variation to neuropsychiatric disorders and also yields insights into the variant genes and the pathways that underlie them.

Neurodegeneration is a progressive loss of neuron function or structure, including death of neurons, and occurs at many different levels of neuronal circuitry. In this thesis I discuss Parkinson’s Disease (PD), the second most common neurodegenerative disease (NDD). PD is a devastating progressive NDD often with delayed diagnosis due to detection methods that depend on the appearance of visible motor symptoms. By the time cardinal symptoms manifest, 60 to 80 percent or more of the dopamine-producing cells in the substantia nigra are irreversibly lost. Although there is currently no cure, earlier detection would be highly beneficial to manage treatment and track disease progression. However, today’s clinical diagnosis methods are limited to subjective evaluations and observation. Onset, symptoms and progression significantly vary from patient to patient across stages and subtypes that exceed the scope of a standardized diagnosis. The goal of this thesis is to provide the basis of a more general approach to study the brain, investigating early detection method for NDD with focus on PD. It details the preliminary development, testing and validation of tools and methods to objectively quantify and extrapolate motor and non-motor features of PD from behavioral and cognitive output during everyday life. Measures of interest are categorized within three domains: the motor system, cognitive function, and brain activity. This thesis describes the initial development of non-intrusive tools and methods to obtain high-resolution movement and speech data from everyday life and feasibility analysis of facial feature extraction and EEG for future integration. I tested and validated a body sensor system and wavelet analysis to measure complex movements and object interaction in everyday living situations. The sensor system was also tested for differentiating between healthy and impaired movements. Engineering and design criteria of the sensor system were tested for usability during everyday life. Cognitive processing was quantified during everyday living tasks with varying loaded conditions to test methods for measuring cognitive function. Everyday speech was analyzed for motor and non-motor correlations related to the severity of the disease. A neural oscillation detection (NOD) algorithm was tested in pain patients and facial expression was analyzed to measure both motor and non-motor aspects of PD. Results showed that the wearable sensor system can measure complex movements during everyday living tasks and demonstrates sensitivity to detect physiological differences between patients and controls. Preliminary engineering design supports clothing integration and development of a smartphone sensor platform for everyday use. Early results from loaded conditions suggest that attentional processing is most affected by cognitive demands and could be developed as a method to detect cognitive decline. Analysis of speech symptoms demonstrates a need to collect higher resolution spontaneous speech from everyday living to measure speech motor and non-motor speech features such as language content. Facial expression classifiers and the NOD algorithm indicated feasibility for future integration with additional validation in PD patients. Thus this thesis describes the initial development of tools and methods towards a more general approach to detecting PD. Measuring speech and movement during everyday life could provide a link between motor and cognitive domains to characterize the earliest detectable features of PD. The approach represents a departure from the current state of detection methods that use single data entities (e.g.one-off imaging procedures), which cannot be easily integrated with other data streams, are time consuming and economically costly. The long-term vision is to develop a non-invasive system to measure and integrate behavioral and cognitive features enabling early detection and progression tracking of degenerative disease.

<p>Across the tree of life, novel genes are thought to be a source of much of the unique behaviors and adaptions between the different taxa. This is especially true in the social insects where novel genes are proposed to contribute to novel social behaviors. In the honey bee (Apis mellifera L.), a group of novel genes called the major royal jelly proteins (MRJPs) are proposed to be important to the expression of novel social behaviors, particularly those related to nursing versus foraging tasks. Unfortunately, identifying the functional role of novel genes is often not possible due to a lack of functional genomic tools in non-model species such as the honey bee. Here I have developed a novel ectopic expression system in Drosophila melanogaster and used it to elucidate how the MRJPs contribute to behavioral and transcriptional changes in the insect brain. I found that the MRJPs regulated the expression of hundreds of genes in Drosophila, and these overlap with genes regulated differentially between nursing and foraging honey bees. Furthermore, I found that MRJP expression impairs or negatively regulated phototaxis. My results demonstrate the MRJPs play a role in behavioral plasticity and highlight that the MRJPs may have a much larger role in the nurse-forager transition than previously thought.</p>

Spinal muscular atrophy (SMA) and Charcot-Marie-Tooth disease type 2D (CMT2D)/distal SMA type V (dSMAV) are two incurable neuromuscular disorders that predominantly manifest during childhood and adolescence. Both conditions are caused by mutations in widely and constitutively expressed genes that encode proteins with essential housekeeping functions, yet display specific lower motor neuron pathology. SMA results from recessive inactivating mutations in the survival motor neuron 1 (SMN1) gene, while CMT2D/dSMAV manifests due to dominant point mutations in the glycyl-tRNA synthetase (GlyRS) gene, GARS. Using a number of different model systems, ranging from Caenorhabditis elegans to the mouse, this thesis aimed to identify potential novel therapeutic compounds for SMA, and to increase our understanding of the mechanisms underlying both diseases. I characterised a novel C. elegans allele, which possesses a point mutation in the worm SMN1 orthologue, smn-1, and showed its potential for large-scale screening by highlighting 4-aminopyridine in a screen for compounds able to improve the mutant motility defect. Previously, the gene encoding three isoforms of chondrolectin (Chodl) was shown to be alternatively spliced in the spinal cord of SMA mice before disease onset. I performed functional analyses of the three isoforms in neuronal cells with experimentally reduced Smn levels, and determined that the dysregulation of Chodl likely reflects a combination of compensatory mechanism and contributor to pathology, rather than mis-splicing. Finally, working with two Gars mutant mice and a new Drosophila model, I have implicated semaphorin-plexin pathways and axonal guidance in the GlyRS toxic gain-of-function disease mechanism of CMT2D/dSMAV.

In this thesis I apply new sequencing technologies and analytical methods derived from genomics and computer science to the neuroanatomy of gene expression. The first project explores characteristics of gene expression across adult neocortical layers in a representative mammal – the mouse. Amongst the thousands of genes and transcripts differentially expressed across layers, I found common functional characteristics of genes that define certain layers, candidate cases of isoform switching, and over a thousand apparent long intergenic non-coding RNA transcripts. The second project compares patterns of gene expression in the structurally diverged adult derivatives of the pallium in mice and chickens. Overall, gene expression levels were moderately correlated between the two species. While expression patterns of ‘marker’ genes were only poorly conserved in these regions, there nevertheless was significant conservation of cross-species marker genes for homologous structures, cell types and functionally analogous regions. Many aspects of these data from both projects can now be easily browsed and searched from custom-built web interfaces. In addition to generating unprecedented genome-wide resources for the neuroscience community to explore the functional and structural dimensions of gene expression amongst different pallial regions in mammals and birds, this work also provides new insights into the widespread evolutionary shuffling of adult marker gene expression.